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WO1992000324A1 - Novel protein capable of causing differential growth of cell and production thereof by genetic engineering technology - Google Patents

Novel protein capable of causing differential growth of cell and production thereof by genetic engineering technology Download PDF

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Publication number
WO1992000324A1
WO1992000324A1 PCT/JP1991/000871 JP9100871W WO9200324A1 WO 1992000324 A1 WO1992000324 A1 WO 1992000324A1 JP 9100871 W JP9100871 W JP 9100871W WO 9200324 A1 WO9200324 A1 WO 9200324A1
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French (fr)
Japanese (ja)
Inventor
Tamotsu Hashimoto
Ken-Ichi Tezuka
Masayoshi Kumegawa
Sunao Takeshita
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Sanofi Aventis KK
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Hoechst Japan Ltd
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Priority claimed from JP25681090A external-priority patent/JPH04117398A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel protein having an ability to form osteocyte differentiation and brain cell differentiation, DNA encoding the same, and a method for producing the protein by a genetic engineering method.
  • Kad 0 matsu et al. (Kadomatsueta 1.1988, Biom. Biolys. Res.Comniun, Vo.151, Pages 1312-1318) are specific during the early development of mouse embryonic teratocarcinoma cells.
  • TGF Transforming growth factor (activator)-indicates that it is a hormone involved in cell growth and differentiation, such as a protein belonging to ⁇ -f ⁇ -milli.
  • BMP Bone Morphogenic Protein
  • Osteoporosis and dementia as senile diseases have recently become a serious social problem in industrialized countries, and the number of patients is also increasing.
  • genes and their products that are specifically expressed in osteogenic and brain cells have the potential to open new avenues for their treatment.
  • the present inventors isolated a cDNA clone specifically expressed in a cell line MC3T3E1 having a property similar to that of a mouse calvaria-derived osteogenic cell (osteoplast) and isolated the same. We have found that DNA clones code for new proteins. The present inventors further used the cDNA encoding the novel protein derived from this mouse as a probe, and obtained the human-derived protein from a cDNA library prepared from human cells. CDNA clones to be cloned were also identified.
  • the protein obtained by the present invention is a protein consisting of 168 amino acids belonging to a family of a kind of cell differentiation or growth factor called MK family.
  • the mRNA encoding this protein is not expressed in mouse kidney cells, spleen cells, thymocytes, liver cells, lung cells, and skeletal muscle cells, but is strong in MC3T3E1 cells and in brain cells. It is weakly expressed in NIH3T3 cells.
  • the present invention first provides an amino acid residue represented by the following sequence [I]: It consists of a protein having a group.
  • a * 1 is S er force
  • G 1 n
  • a * 2 is S er or A 1 a
  • a * 3 is Le eu or P he ⁇
  • a * 4 is Asp or G lu.
  • a preferred example of the protein of the present invention is a protein named mouse OSF-1 or human 0SF-1 represented by the following sequence [I-I] or [I- ⁇ ]. [ ⁇ -I]
  • the present invention further comprises a DNA encoding the above sequence [I].
  • a part of the amino acid residue in the sequence [I] can be modified by deleting, replacing or adding the amino acid without significantly changing the properties of the protein. Further, not only DNA encoding the entire sequence [I] or the modified sequence [I] but also DNA encoding the portion thereof are included in the scope of the present invention.
  • the present invention also relates to an expression vector, which comprises a DNA encoding the above-mentioned protein and an appropriate gene promoter linked to its upstream end in the correct direction, for example, gene expression in animal cells. It is characterized by incorporating the early promoter of the T antigen of SV4G virus, which is commonly used for, and having DNA fragments necessary for the expression of other exogenous genes.
  • the present invention relates to a method for producing the protein represented by the above-mentioned sequence [I], which comprises transforming a host cell with the expression vector of the present invention and culturing the transformant. It is characterized by producing the protein.
  • the present invention relates to a protein obtained by culturing the transformant.
  • protein refers to a protein having the entire amino acid sequence of the above [I], a protein having the entire amino acid sequence partially modified by deletion, substitution, or insertion; It consists of an array part.
  • Example of DNA encoding 168 amino acid residues according to the present invention In example in Chinese hamster ovary cells (CHO) animal cells, such as, for example, by expressing linked downstream of the initial promoter of T antigen S V40 virus are properly even all 1 68 ⁇ Mi Amino Acids that It is possible to secrete and produce a protein having a partial sequence outside the host cell. In addition, since this protein does not have a site for modifying a sugar chain, it can be produced using a microorganism such as yeast or Escherichia coli. The protein thus produced, like other proteins belonging to the MK family, has the effect of promoting the growth and differentiation of osteoblasts and brain nerve cells.
  • CHO Chinese hamster ovary cells
  • the cDNA is used as a probe to isolate human alleles from the cDNA library of human osteoblasts and brain nerve cells, or cell lines established from these tumor lines. It is also possible.
  • the protein according to the present invention is a protein specifically and strongly expressed in the cDNA library of MC3T3E1 cells, which are osteoblast-like cells established from mouse calvarial cells, Was selected in comparison to the sexual fibroblast cell line, NIH3T3.
  • cDNA encoding human OSF-1 was also selected from the cDNA library of humans using the cDNA encoding mouse SF-1 obtained as a probe.
  • the nucleotide sequence of those cDNAs is represented by [ ⁇ ].
  • mouse brain, muscle, kidney, liver, spleen, thymus was confirmed by Northern hybridization, expression was observed only in brain cells except for MC3T3E1 cells.
  • 0 S F-1 is a novel protein having the following properties.
  • MK-1 MGF REGT-CGAQT Q VHC VPCN WK EFGADC
  • OSF-1 YQFQAWGECD LNTA1KT TG SLKRA1HNAD CQKTVT S P CGK1T P PQ
  • MK-1 Y FESWGACD GSTGTKARQG TL KARYTAQ CQET RVTKP CTSKTKS T
  • OSF-1 AESKKKK EG KKQEKMLD
  • MK-1 is known to be a secreted protein (or polypeptide), and has been suggested to be a differentiation factor for empironic carcinoma cells under the control of retinoic acid (Mineko Tomomura, Kenji Kadomatsu, Takashi Muramatsu, Biochemistry, 1989, Voi.61, ⁇ 9, Page 1040), and OSF-1 are very similar in structure to MK-1 except that their tissue specificities are osteoblasts and brain cells.
  • the protein of the present invention may be a protein containing all of the amino acid residues of OSF-11 or a partially substituted amino acid residue of OSF-1 having substantially the same activity as OSF-1. Including.
  • the protein of the present invention can be secreted as a protein consisting of the sequence [I]. Judging from the similarity with MK-1, it can be secreted as a protein with a molecular weight of around 90 after being cleaved from Gin (Q) at position ⁇ to Arg (R) at position 84. There is. Therefore, the protein of the present invention includes a protein consisting of the part of the sequence [I] and a partial amino acid residue having substantially the same activity as the protein.
  • the DNA encoding the protein according to the present invention has the sequence
  • a typical sequence of DNA according to the present invention is the following sequence [ ⁇ ].
  • U is A or G
  • Y is C or T
  • X is G or T
  • Z is A force
  • C and V are G or C
  • W is T or A.
  • DNA encoding mouse OSF-1 and human OSF-1 are represented by the following sequences [ ⁇ -I] and [ ⁇ - ⁇ ].
  • DNA encoding the protein according to the present invention includes those having the nucleotide sequence of the above-mentioned sequence [ ⁇ ] and degenerate isomers thereof.
  • degenerate isomer shall mean DNA which has the same nucleotide sequence and encodes the same protein except that codons having a degenerate relationship are used.
  • one means for obtaining the DNA is to convert nucleic acid. It is to produce according to the method of chemical synthesis.
  • the DNA encoding mouse OSF-1 according to the present invention can be obtained from a mouse calvarial osteoblast MC3T3E1 (Kodama et al., 1981, Jpn. J. Oia 1 Biol., Vo.23, (Pages 899-901) Cells were seeded in 2.2 ⁇ 10 5 cells / plate in 40 Petri dishes with em diameter and the medium was changed appropriately at 9 ° C for about 9 days.After culturing, the cells became 8 ⁇ 1G 7 cells.
  • Maniatis et al. Maniatis et al., 1982, Molecular Cloning: A Laoratory Manual, Cold Spring Harbor Laboratory
  • After preparing total RNA purify mRNA, synthesize cDNA, clone It can also be manufactured by conversion.
  • the cDNA derived from this mouse can also be produced as a probe from the cDNA library of another animal.
  • the OSF-1 protein can be produced using Escherichia coli, yeast, or animal cells as the host, using the corresponding promoter. As a specific method, the method described in Maniatis et al. (Supra) can be used.
  • Restriction enzymes were obtained from New England Biolabs, USA, and Takara Shuzo, and were used according to the manufacturer's instructions unless otherwise noted.
  • Exit Delane kit for sequence cloning vector 3-ter PUC 118, T4 DNA ligase and bacterial phosphatase were obtained from Takara Shuzo and used according to the manufacturer's instructions.
  • c DNA synthesis system ⁇ Plus was obtained from Amersham, UK, and used according to the manufacturer's instructions.
  • AMV reverse transcriptase, mRNA purification kit, and Ec QRI-Not I adapter were obtained from Pharmacia, Sweden, and used according to the manufacturer's instructions. .
  • Random primed DNA labeling kits were obtained from Behringer Mannheim Yamanouchi and used according to the manufacturer's instructions.
  • i gtio cloning vector and invitrono zero packaging kit "Gigapack Gold” were obtained from Stratagene, USA, (Stratagene) and the manufacturer's instructions. Used according to. Transformation of Escherichia coli (E. coli) and ligation of DNA were performed according to the method described by Mania tis et al.
  • FIG. 1 shows the structure of the plasmid pMC031.
  • the white band indicates the cDNA insertion site of mouse OSF-1.
  • the arrow indicates the ampicillin resistance gene.
  • the black circles indicate the starting point of plasmid replication.
  • FIG. 2 shows the structure of the plasmid PMCG31 and the cDNA inserts of the 10 deletion mutants, and the region in which the base sequence was determined.
  • the solid line indicates the cDNA insert contained in each plasmid. Arrows indicate the region and direction in which the base sequence was determined.
  • FIG. 3 shows the entire nucleotide sequence of the cDNA insertion site of pMCl and the amino acid sequence of mouse 0 S F-1 deduced from the nucleotide sequence.
  • the underline indicates the portion derived from the EcoRI-NotI adapter. *** indicates a transcription termination codon.
  • FIG. 4 shows examination of tissue-specific expression of the 0SF-1 gene by Northern plotting.
  • Each lane is as follows. 1; thymus, 2; spleen, 3; brain, 4; kidney, 5; liver, 6; lung, 7; skeletal muscle, 8;
  • FIG. 5 shows the structure of plasmid PHSG757.
  • the white band indicates the poly A signal
  • the white band with the arrow indicates the SV40T antigen early promoter
  • the arrow indicates the chloramphenicol resistance gene.
  • the black circle indicates the origin of plasmid replication.
  • FIG. 6 shows the entire nucleotide sequence of the cDNA fragment of pHBR1 and the amino acid sequence of human OSF-1 deduced from the nucleotide sequence.
  • Example 1 Mouse calvaria-derived osteoblast-like cell line
  • RNA was prepared from 1.5-cells (Kodama et al., 1981, Jpn. J. Oral Biol., 23, 899-901) by the guanidine method (Mani at is, et al., Supra), and then the total RNA was prepared.
  • mRNA was prepared using an mRNA purification kit. Using the prepared mRNA as type I, a cDNA synthesis system plus (Amersham)
  • Double-stranded cDNA was synthesized. First, Ec0RI-N01I adapter-1 (Pharmacia) was ligated to the synthesized double-stranded cDNA using T4 DNA ligase, and then digested with EcoRI; Cloning vectors (Stratagene) were ligated using T4 DNA ligase.
  • the i gtlO c DNA was linked to a, b down bi-DOO be sampled La evening Gee down Russia Nono 0 Tsu cage packaging kit "Gigapa' Kugoru de (G igapac It Gold)" using a stomach down bi collected by filtration ( The cells were packaged in a lambda phage in vitro and stored in SM buffer (Mani at is, et al., supra). This was transformed into E. coli C600 strain (National Institute of Preventive Health Gene Bank).
  • a cDNA library was generated at a frequency of approximately 1.4 ⁇ 10 7 primary phage plaques per gram of cDNA.
  • Example 2 Difference library of cDNA library derived from MC3T3E1 cells with NIH3T3 cells (ATCC CRL 1658). ) To select clones specific to MC3T3E1 cells and to clone the plasmid vector pUCIU. MC 3 T 3 E 1 cells own mRNA and MC 3 T 3 E
  • NIH 3T3 cells mouse fibroblast cell line NIH 3T3 cells (ATCC CRL 1658) prepared in the same manner as 1 cells as type III, the cells were treated with AMV Linokin Strand Scryptase (Pharmacia), respectively.
  • a radioactive cDNA probe was synthesized.
  • cDNA library derived from MC3T3E1 cells about 1.5 ⁇ 10 4 cells were subjected to the plaque-hybridization method (Maniatis, et al.) Using each radioactive cDNA probe. According to the above, screening was performed sequentially to obtain 78 clones specifically hybridizing to a radioactive cDNA probe derived from MC3T3E1 cells.
  • each phage DNA was purified from the lysate of these phage clones by phenol treatment followed by ethanol precipitation (Maniat is et al .; supra), digested with EcoRI, separated by agarose gel electrophoresis, c Only the DNA insert was purified (Maniatis et al., supra).
  • radioactive probes derived from each clone were prepared using a random primed DNA labeling kit (Berlingermanheim Yamanouchi).
  • mRNA 0.3 ⁇ of MC3T3E1 cells and NIH3T3 cells was separated by formaldehyde denaturing agarose gel electrophoresis (Mani at is, et al., Supra), and then separated by a nylon membrane (BYODYNE , Pall Bio Support, USA) (aniatis ⁇ , supra) 0
  • cDNA insert of this Phage clone MC031 was digested with the restriction enzyme EcoRI, and the 5'-terminal phosphoric acid was removed by treatment with bacterial alkaline phosphatase (Takara Shuzo). It was ligated with PUC118 (Takara Shuzo) using T4 DNA ligase to obtain plasmid pMCl.
  • Fig. 1 shows the structure.
  • pMC031-1 After digesting pMC031 with the restriction enzymes SphI and BamHI, ten deletion mitants were prepared using a kilosequence deletion kit (Takara Shuzo). These are referred to as pMC031-1, pMC031-2, pMC031-3,..., PMC0310 (FIG. 2). Applied Biosystems, Inc., USA, (Applied Biosystems), using an automatic sequence analyzer (Model 370A), pMC031, and their displacement mutators. Approximately 300 base pairs were determined from the Hind site side of the cDNA insertion site. FIG. 3 shows the entire nucleotide sequence of the resulting cDNA insert and the amino acid sequence deduced therefrom. The protein presumed to be coded by this gene is called mouse OSF-1.
  • Example 4 Examination of Tissue-Specific Expression of Mouse OSF-1 Tissue-specific expression of mouse 0SF-1 gene was examined by Northern blotting.
  • the cDNA insert of pMCl was digested with EcoRI and purified by agarose gel electrophoresis (Maniatis et al., Supra), and this was used as a substrate for randomization in the Behringermannheim Yamanouchi. Radioactive probes were prepared using the primed DNA labeling kit.
  • FIG. 6 shows the entire nucleotide sequence of the resulting cDNA insert and the amino acid sequence deduced therefrom.
  • SF-1 was produced using the method described in JP-A-63-7288 for efficiently producing a protein in animal cells using the amplified gene. ⁇ ⁇
  • cDNA encoding the same protein must be transferred to cassette vector p HSG (National Institute of Health).
  • Insertion number VEH 7 was inserted between the SV40 virus T antigen early promoter and the polymerase II signal as follows. The EcoRI site upstream of the SV40T antigen early promoter of pHSG747 was eliminated by digestion with EcoRI, blunting of the terminal with the Klenow fragment (Takara Shuzo), and subsequent recombination with ligase. Was.
  • the single Pst1 site between the SV40T antigen early promoter and the polyA signal sequence was replaced with a synthetic EcoRI linker (oligonucleotide) (Takara Shuzo), and a single EcoR at the same position. It has an I site.
  • a plasmid pHSG757 was constructed for amplification of the in vitro gene (Fig. 5).
  • An OSF-1 cDNA fragment sandwiched between the EcoRI sites was inserted in the EcoRI site in the forward direction to obtain plasmid pOFl.
  • BstXI By digesting this plasmid with the restriction enzyme BstXI, an expression unit DNA fragment of 0SF-1 having asymmetric sticky ends at both ends as described below can be obtained.
  • plasmid pHSGII National Institute of Health and Prevention Genebank registration number VE9
  • the fragment of [m] obtained in this manner was prepared by digesting the DNA of Cosmid Vector-p HSG 293 (National Institute of Preventive Health, Gene Bank registration number VE 046) with BstXI digestion as follows: And 20: 1, and ligated to tandem by T4 ligase according to the method described in the above-mentioned JP-A-sho.
  • OSF-1 cDNA fragment was used as a probe by the northern blotting method (Maniais et al., Supra). A strain that expressed the same gene with high efficiency was obtained.
  • the selected OSF-1 producing strain contains 10% fetal bovine serum-MEM medium (GIBC 0) And OSF-1 was detected in the culture supernatant.
  • novel protein having osteocyte differentiation-forming ability, DNA encoding the same, and a method for producing the protein by a genetic engineering method.
  • the novel protein provided by the present invention has the potential to be used for the treatment of osteoporosis and dementia as senile diseases.

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Abstract

A protein capable of causing differential growth of osteoblasts and cranial nerve cells originating in mouse calvarial cells, having 168 amino acid residues, and useful for treating and diagnosing osteoporosis and dementia. It is produced by expressing DNA which codes for this protein.

Description

明 細 書 細胞分化形成能を有する新規な蛋白質と その遺伝子工学的製法 技 術 分 野  Description Novel proteins capable of forming cell differentiation and their genetic engineering manufacturing technology

本発明は、 骨細胞分化形成能および脳細胞分化形成能を 有する新規な蛋白質、 それをコー ドする D N Aおよびその 遺伝子工学的製法によるこの蛋白質の製造方法に関する。 背 景 技 術  The present invention relates to a novel protein having an ability to form osteocyte differentiation and brain cell differentiation, DNA encoding the same, and a method for producing the protein by a genetic engineering method. Background technology

K a d 0 m a t s u 等 (K a d o m a t s u e t a 1. 1988, B i o c e m. Bi oply s. Res. Comniun, Vo .151, Pages 1312-1318) はマ ウスの胚性奇形癌腫細胞の初期発生の過程で特異的に発現 する高塩基性の リ ジン残基に富む MK 1 と名付けた分子量 9， 971ダル ト ンのポ リペプチ ドをコー ドする遺伝子を見つ けた。 彼 ら は （ K a d 0 m a t s u e t a I. 1990, J. o f Ce l l Biol. , Vo .110, Pages 607 - 616 ) 更にこのペプチ ドが分 泌性のものであるこ とを明らかにし、 T G F (Trans forming growth f actor) - βフ τ ミ リ ーに属する蛋白質のよう に細胞の成長分化に関与するホルモンであることを示して いる。 T G F - βつ マ ミ リ ーの蛋白質は最近 B M P (Bone Mo rphogen i c Protein)と呼ばれる一連の骨形成に関与する 蛋白質の存在が報告されており骨折治療への応用が期待 されてい る （た とえば Wozney e t a 1. 1988 , Sc i ence, Voi.242, Pages 1528 - 1534 ) 。 また、 脳細胞に由来する蛋 白質性の成長因子と しては、 N G F (Nerve grovth factor) 等が報告されており、 その医学領域での応用が期待されて いる。 Kad 0 matsu et al. (Kadomatsueta 1.1988, Biom. Biolys. Res.Comniun, Vo.151, Pages 1312-1318) are specific during the early development of mouse embryonic teratocarcinoma cells. A gene encoding a polypeptide with a molecular weight of 9,971 daltons, named MK1, which is rich in highly basic lysine residues that was expressed in the yeast, was found. They further (Kad 0 matsueta I. 1990, J. of Cell Biol., Vo. 110, Pages 607-616) further revealed that this peptide was secretory, and that TGF (Transforming growth factor (activator)-indicates that it is a hormone involved in cell growth and differentiation, such as a protein belonging to β-f τ-milli. Recently, it has been reported that the TGF-β family protein has a series of proteins involved in bone formation called BMP (Bone Morphogenic Protein), and is expected to be applied to the treatment of fractures. For example, Wozney eta 1. 1988, Science, Voi.242, Pages 1528-1534). In addition, proteins derived from brain cells NGF (Nerve grovth factor) has been reported as a white matter growth factor, and its application in the medical field is expected.

老人性の疾患と しての骨粗ショ ゥ症及び痴呆症は近年 益々先進工業国において深刻な社会問題となりつつあり、 その患者数も増加の傾向にある。 従って骨形成細胞及び脳 細胞に特異的に発現される遺伝子及びその生産物はそれら の治療に新しい道を開く可能性を秘めている。  Osteoporosis and dementia as senile diseases have recently become a serious social problem in industrialized countries, and the number of patients is also increasing. Thus, genes and their products that are specifically expressed in osteogenic and brain cells have the potential to open new avenues for their treatment.

発 明 の 開示 Disclosure of the invention

本発明者は、 マウス頭蓋冠由来の骨形成細胞 （ォステ オプラス ト） 様の性質を持つ細胞株 M C 3 T 3 E 1 におい て特異的に発現 している c D N Aク ロー ンを分離し同 c D N Aク ローンが新規な蛋白質をコ一 ドすることを見い だした。 本発明者はさ らに、' このマウス由来の新規な蛋白 質をコー ドする c D N Aをプローブと して用い、 ヒ ト細胞 より作成した c D N Aライブラ リーからヒ ト由来の本蛋白 質をコー ドする c D N Aク ローンをも分離同定した。  The present inventors isolated a cDNA clone specifically expressed in a cell line MC3T3E1 having a property similar to that of a mouse calvaria-derived osteogenic cell (osteoplast) and isolated the same. We have found that DNA clones code for new proteins. The present inventors further used the cDNA encoding the novel protein derived from this mouse as a probe, and obtained the human-derived protein from a cDNA library prepared from human cells. CDNA clones to be cloned were also identified.

本発明で得られる蛋白質は MKフ ァ ミ リ ーと呼ばれる一 種の細胞分化または成長因子のフア ミ リ ーに属するァ ミ ノ 酸 168個からなる蛋白質である。 この蛋白質をコー ドする m R N Aはマウスの腎細胞、 脾臓細胞、 胸腺細胞、 肝臓細 胞、 肺細胞、 骨格筋細胞では発現せず、 M C 3 T 3 E 1細 胞で強く、 また脳細胞と N I H 3 T 3細胞では弱く発現す 。  The protein obtained by the present invention is a protein consisting of 168 amino acids belonging to a family of a kind of cell differentiation or growth factor called MK family. The mRNA encoding this protein is not expressed in mouse kidney cells, spleen cells, thymocytes, liver cells, lung cells, and skeletal muscle cells, but is strong in MC3T3E1 cells and in brain cells. It is weakly expressed in NIH3T3 cells.

本発明はまず、 下記の配列 〔 I〕 で表されるァ ミ ノ酸残 基を有する蛋白質からなるものである。 The present invention first provides an amino acid residue represented by the following sequence [I]: It consists of a protein having a group.

Met-A*l-A*2-Gln-Gln-Tyr-Gln-Gln-Gln-Arg-Arg-Lys-Phe-Ala-Ala-

Figure imgf000005_0001
Met-A * lA * 2-Gln-Gln-Tyr-Gln-Gln-Gln-Arg-Arg-Lys-Phe-Ala-Ala-
Figure imgf000005_0001

Glu-AJLa-Gly-Lys-Lys-Glu-Lys-Pro-Glu-Lys-Lys -Val-Lys-Lys-Ser- Glu-AJLa-Gly-Lys-Lys-Glu-Lys-Pro-Glu-Lys-Lys -Val-Lys-Lys-Ser-

Asp-Cys-Gly-Glu-Trp-Gln-Trp-Ser-Val-Cys-Val-Pro-Thr-Ser-Gly-- Asp - Cys - Gly— Leu - GIY - Thr - Arg - Glu - Gly— Thr - Arg- Thr - Gly- Ala-Glu— Cvs-Lys-Gln-Thr-Met—Lvs—Thr-Glxi-A g-Cys-Lys-Ile—Pro-Cys-Asii— Trp-Lys-Lys-Gln-Phe-Gly-Ala-Glu-Cys-Lys-Tyr-Gln-Phe-Gln-Ala- Trp-Gly-Glu-- Cys -Asp-Leu -Asn- T r-Ala-Leu-Lvs -Thr-Arg- Thr-Gly- Ser-Leu— Lvs— Arg— Ala - Leu- His - Asn - Ala - A*4 - Cys - Gln-Lys - Thr - Val— Thr-Ile-Ser-Lys-Pro-Cvs-Gly-Lys-Leu-Thr-Lys-Pro-Lvs-Pro-Glii- Ala - Glu— Sear - Lys - Lys - Lys— Iys - Lys - Glu— Gly - Lys—Lys—Gln—Glu—Iiys - Met— Leu - Asp Asp-Cys-Gly-Glu-Trp-Gln-Trp-Ser-Val-Cys-Val-Pro-Thr-Ser-Gly-- Asp-Cys-Gly— Leu-GIY-Thr-Arg-Glu-Gly— Thr -Arg- Thr-Gly- Ala-Glu— Cvs-Lys-Gln-Thr-Met—Lvs—Thr-Glxi-A g-Cys-Lys-Ile—Pro-Cys-Asii— Trp-Lys-Lys-Gln- Phe-Gly-Ala-Glu-Cys-Lys-Tyr-Gln-Phe-Gln-Ala- Trp-Gly-Glu-- Cys -Asp-Leu -Asn-Tr-Ala-Leu-Lvs -Thr-Arg- Thr-Gly- Ser-Leu— Lvs— Arg— Ala-Leu- His-Asn-Ala-A * 4-Cys-Gln-Lys-Thr-Val— Thr-Ile-Ser-Lys-Pro-Cvs-Gly- Lys-Leu-Thr-Lys-Pro-Lvs-Pro-Glii-Ala-Glu—Sear-Lys-Lys-Lys—Iys-Lys-Glu—Gly—Lys—Lys—Gln—Glu—Iiys-Met—Leu— Asp

〔 I 〕 [I]

配列中、 A * 1 は S e r力、 G 1 n、 A * 2 は S e rか A 1 a、 A * 3は L euか P heヽ A * 4は Aspか G lu、 である。  In the sequence, A * 1 is S er force, G 1 n, A * 2 is S er or A 1 a, A * 3 is Le eu or P he ヽ A * 4 is Asp or G lu.

本発明の蛋白質の好適な例は下記の配列 〔 I - I〕 又は 〔 I - Π〕 で表わ さ れる マ ウ ス O S F - 1 又は ヒ ト 0 S F - 1 と名付けられる蛋白質である。 〔Π - I〕 A preferred example of the protein of the present invention is a protein named mouse OSF-1 or human 0SF-1 represented by the following sequence [I-I] or [I-Π]. [Π-I]

— s« i— rr )— て3— SAT;— sAq;— Λて 一 r\i[£i一 ε τ;— SAT;— s τ:— εΑτ:— ·^Ί—ュ 3S— nて 3— — S «i— rr) — te3— SAT; — sAq; — Λtei r \ i [£ i ε τ; — SAT; — s τ: — εΑτ: — · ^ Ί—Ί 3S— nte 3—

— IXて 3— or — s 一 oュ d— s T ュ xii— ST;— S x— て)— s っー 0ュ3— SA —ュ as— 3てエー: z j; —て B v— :i

Figure imgf000006_0001
— IX te 3— or — s one o d— s T te xii— ST; — S x—te) — s te 0 te 3— SA — te as— 3 te: zj; — te B v— : i
Figure imgf000006_0001

— Aて 3—ュ i— β^Υ-ユ^ !«— S^T:— — Bて :r c— χιε·γ— ΠΒΤ:— <3 — s :)— riて)一 — d ^ 一 *ε — tiて 3— — IIて )— ijQ— S T:— s^ —ひて 3— " eT — T)—3 <i_uTS—sArI—SifI-<i:r — A then 3 — i — β ^ Υ-yu ^! «— S ^ T: — — B: rc— χιε · γ— ΠΒΤ: — <3 — s:) — rite) one — d ^ one * ε — ti then 3— — II) — ijQ— ST: — s ^ —h 3— " e T — T) —3 <i_ u TS— s A r I— Sif I- <i: r

一ひ — s 0— oュ d— 3てエー s Ί一 s^O— 5ュ Y—ひて —ユ^ I；一 SAT;—: an—ュ ェ— uて 3— s Ί一 SAQ 一 nて 3— eて γ— て>—ュ q£— β^τ¾·—ュ1^ Q— て 3— て £)— β Γ¾"— ^て 3— て )-s l一 ds^f 一 Λて 3—ュ as—ュ o:r<j—て Λ— s^ —て —ュ as— <ir«L— IIて 3— nて 3—^て 3— つ一 ds Ichi — s 0 — o d — 3 te s Ί s ^ O — 5 5 Y — te — yu ^ I; i SAT; —: an — e — u te 3 — s Ί ichi SAQ ichi n Te Te 3- e Te γ-> - Interview q £ - β ^ τ¾ · - Interview 1 ^ Q- Te 3-Te £) - β Γ ¾ "- ^ Te 3-Te) -sl one ds ^ f one Λte 3 ュ as ュ o: r <j— Λ Λ s ^ — te — as as— <ir «L— II te 3 n te 3 ^ te 3 d

一: res— — SAT;—て — s q:— s 1!一 て 3— O:T(J— s q:— IIて £— S ^I-SAT:— て 3— T — riて f) One: res— — SAT; —te — sq: — s 1 ! One three— O: T (J— sq: — IIte £ — S ^ I-SAT: —te 3— T—rite f)

Si — "e - tii— ds"¾;—て ΒΛ— Βχ — ST — τΐ3*ι— 3てェ一 a <2— 3てェ

Figure imgf000006_0002
Si — "e-tii— ds"¾; —te ΒΛ— Βχ — ST — τΐ3 * ι— 3te1 a <2—3te
Figure imgf000006_0002

— て ¾"-3t{ — s >i— 一 uて )一 uて —ユ^ϋ一 uて 5)-111：3— —11て 3- ^ H — T ¾ "-3t {— s> i— one u t) one u t — u ^ ϋ1 u t 5) -111: 3— — 11 t 3- ^ H

c I - n  c I-n

dE -ns^[- .SH dE -ns ^ [-.SH

―

Figure imgf000006_0003
Οϊ 一 て€)—o:r<i— s i—oors— s q:— !Oi—nsT:— SAT;— て £)一 — oora-s Ί一： rss_3てェ-: rqji -て SA—ュ qi-s q;— UT )— s^o— di -BTY-us -STH-naT!-BX -jS Y-sAi-na^i-rss —ュ q ー ·5 ¾·-;τΐί£-ε·<¾— ·η3τ;-·εて γ—: oiJi— τιε¾·— naT:_<iEY-S i )— nて 3— て ―
Figure imgf000006_0003
Οϊ one €) —o: r <i— si—oors— sq: —! Oi—nsT: — SAT; — te £) one — oora-s Ίichi: rss_3te-: rqji-te SA qi-s q; — UT) — s ^ o— di -BTY-us -STH-naT! -BX -jS Y-sAi-na ^ i-rss — ュ q · 5 ¾ ·-; τΐί £ -ε · <¾— · η3τ;-· εte γ—: oiJi— τιε¾ · — naT: _ <iEY-S i) — nte 3—te

— tiて 3— 3q<j— uて 3—ュ £一 SAT;— 5^コ一 tiて 3— Β ¾·— て £)一 — tiTf一 Sirr— s >l-d^ェ  — Ti then 3— 3q <j— u then 3— ££ SAT; — 5 ^ 1 ti and 3— Β ¾ · — ££ 1— tiTf-1 Sirr— s> l-d ^ ェ

- USY - 3 コ_0ュ<3： - 3てエ- s_<¾- ε - £ュ¾： - Iて 3 -: τςϋ - s_<¾— eH-ェ - て 3 - s 一 s^o 5 -tiて 3-Bて γ— Λて s-rcqi-S y—ュ q -^て 一 nて 3-·5:τ¾·-ュ qェ — nsi- て 3— s o-ds^f — て 3—ュ as—ュ：！ 一 0ュ<1— TSA— て —r3S_ir;—iて 3_<i ー nて て 3— s d_ds¾" -■ ss- S iT:-

Figure imgf000006_0004
-USY-3 _0 <3:-3-s_ <¾-ε-£ ¾:-I 3-: τςϋ-s_ <¾— eH-e-3-s one s ^ o 5 -ti and 3-B and γ- and s-rcqi-S y- and q- ^ and 1 n and 3- · 5: τ¾- and q- — nsi- and 3— s o-ds ^ f — And 3—as :! 1 0 <1— TSA— and —r3S_ir; —i and 3_ <i-n and 3— s d_ds¾ "-■ ss- S iT:-
Figure imgf000006_0004

一 Β"ΓΥ—ュ q£— εγ—て ΒΛ— ·ε — STY— Π3Ί— 3てェ一 sti<2— 3てェ _na«i— ST — nsq;— 3 3— ST 1 Β "ΓΥ- ュ q £ — εγ— て ΒΛ— · ε — STY— Π3Ί— 3 ェ sti <2— 3 ェ _na« i— ST — nsq; — 3 3— ST

- STS"—Bて γ一 sqa—s - ·ο:ι - ·5· ¾·—ΐχχ3 - 一 trif)一ュ 一∑T£) - -： - ccss- sH -S TS "—B t γi sqa—s-· ο: ι-· 5 · ¾ · —ΐχχ3-one trif) 1∑1∑T £)--:-ccss-sH

W£00/t6 OAVW £ 00 / t6 OAV

U800/l6df/IOd 本発明はさ らに上記配列 〔 I〕 をコー ドする D N Aから なるものである。 U800 / l6df / IOd The present invention further comprises a DNA encoding the above sequence [I].

配列 〔 I〕 におけるア ミ ノ酸残基の一部分は、 蛋白質の 性質を大き く変えない範囲においてア ミ ノ酸を削除し、 置 換し又は付加することによって修飾することができる。 ま た配列 〔 I〕 も しく は修飾された配列 〔 I〕 の全体をコ一 ドする D N Aのみならずその部分をコ一 ドする D N Aも本 発明の範囲に含まれる。  A part of the amino acid residue in the sequence [I] can be modified by deleting, replacing or adding the amino acid without significantly changing the properties of the protein. Further, not only DNA encoding the entire sequence [I] or the modified sequence [I] but also DNA encoding the portion thereof are included in the scope of the present invention.

また本発明は、 発現ベク タ ーに関するものであり、 上記 の蛋白質をコー ドする D N Aと、 その上流側末端に正しい 方向に連結された適切な遺伝子プロモータ一、 例えば、 動 物細胞での遺伝子発現に良く使われる S V 4Gウィルスの T アンチゲンの初期プロモーターを組込み、 かつ他の外来性 遺伝子の発現に必要な D N A断片をもたせたものを特徴と する ものである。  The present invention also relates to an expression vector, which comprises a DNA encoding the above-mentioned protein and an appropriate gene promoter linked to its upstream end in the correct direction, for example, gene expression in animal cells. It is characterized by incorporating the early promoter of the T antigen of SV4G virus, which is commonly used for, and having DNA fragments necessary for the expression of other exogenous genes.

さ らに本発明は、 上記 〔 I〕 の配列で表される蛋白質の 製造法に関する ものであって、 本発明による発現ベクター で宿主細胞を形質転換し、 該形質転換体を培養することに より該蛋白質を生産するこ とを特徴とする ものである。  Furthermore, the present invention relates to a method for producing the protein represented by the above-mentioned sequence [I], which comprises transforming a host cell with the expression vector of the present invention and culturing the transformant. It is characterized by producing the protein.

さ らに本発明は、 該形質転換体を培養するこ とによって 得られる蛋白質に関する。 こ こでいう蛋白質とは上記 〔 I〕 のア ミ ノ酸配列の全てを有する蛋白質、 ア ミ ノ酸の一部が 削除、 置換、 挿入により修飾された配列の全てを有する蛋 白質、 およびそれらの配列の部分よりなる ものである。  Further, the present invention relates to a protein obtained by culturing the transformant. As used herein, the term "protein" refers to a protein having the entire amino acid sequence of the above [I], a protein having the entire amino acid sequence partially modified by deletion, substitution, or insertion; It consists of an array part.

本発明による 168ア ミ ノ酸残基をコー ドする D N Aを例 えばチャイニーズハムスター卵巣細胞 （C H O) などの動 物細胞中で、 例えば S V40ウィルスの T抗原の初期プロ モーターの下流に連結して発現させることにより、 168ァ ミ ノ酸の全ても しく はその一部の配列を有する蛋白質を宿 主細胞外に分泌生産させることが可能である。 またこの蛋 白質には糖鎖の修飾する部位がないので、 酵母や大腸菌な どの微生物を使って生産することも可能である。 このよう にして生産された蛋白質は MKフア ミ リ ーに属する他の蛋 白質のように骨芽細胞や脳神経細胞の成長分化を促進する 作用を持つ。 したがって、 骨粗ショ ウ症及び痴呆症の治療 剤と して効果が期待される。 また、 この c D N Aをプロ一 ブと してヒ トの骨芽細胞や脳神経細胞、 またはそれらの腫 瘍株から樹立した株細胞の c D NAライブラ リ ーからヒ ト の対立遺伝子を単離することも可能となる。 Example of DNA encoding 168 amino acid residues according to the present invention In example in Chinese hamster ovary cells (CHO) animal cells, such as, for example, by expressing linked downstream of the initial promoter of T antigen S V40 virus are properly even all 1 68 § Mi Amino Acids that It is possible to secrete and produce a protein having a partial sequence outside the host cell. In addition, since this protein does not have a site for modifying a sugar chain, it can be produced using a microorganism such as yeast or Escherichia coli. The protein thus produced, like other proteins belonging to the MK family, has the effect of promoting the growth and differentiation of osteoblasts and brain nerve cells. Therefore, it is expected to be effective as a therapeutic agent for osteoporosis and dementia. The cDNA is used as a probe to isolate human alleles from the cDNA library of human osteoblasts and brain nerve cells, or cell lines established from these tumor lines. It is also possible.

0 S F - 1蛋白質  0 S F-1 protein

本発明による蛋白質は、 マウス頭蓋冠細胞から樹立され た骨芽細胞様細胞である M C 3 T 3 E 1細胞の c D NAラ ィブラ リ ーにおいて特異的に強く発現されるものを、 マウ ス胚性繊維芽細胞株である N I H 3 T 3 と比較対照して選 択された。 さ らにこの得られたマウス〇 S F - 1をコー ド する c D N Aをプローブと してヒ 卜の c D N Aライ ブラ リ ーから ヒ ト O S F - 1をコー ドする c D N Aも選択され た。 それらの c D N Aの塩基配列は 〔Π〕 で表されるもの である。 さ らに、 マウス O S F - 1の組織特異的発現を調 ベるため、 マウスの脳、 筋肉、 腎臓、 肝臓、 脾臓、 胸腺、 肺の各組織での m R N A産生をノーザンハイブリダィゼィ ショ ン法で確認したところ、 MC 3 T 3 E 1細胞以外では 脳細胞でのみ発現が見られた。 The protein according to the present invention is a protein specifically and strongly expressed in the cDNA library of MC3T3E1 cells, which are osteoblast-like cells established from mouse calvarial cells, Was selected in comparison to the sexual fibroblast cell line, NIH3T3. In addition, cDNA encoding human OSF-1 was also selected from the cDNA library of humans using the cDNA encoding mouse SF-1 obtained as a probe. The nucleotide sequence of those cDNAs is represented by [Π]. In addition, to determine the tissue-specific expression of mouse OSF-1, mouse brain, muscle, kidney, liver, spleen, thymus, When mRNA production in each lung tissue was confirmed by Northern hybridization, expression was observed only in brain cells except for MC3T3E1 cells.

0 S F - 1は、 下記の性状を有する新規蛋白質である。 0 S F-1 is a novel protein having the following properties.

① 翻訳開始点であるメ チォニン残基を含め 168ア ミ ノ酸 残基からなり、 その中に Π%〜18%の高含有率でリ ジン 残基を含み塩基性親水性の高い蛋白質である。 (1) Consists of 168 amino acid residues including the methionine residue, which is the translation initiation point, and contains a high content of 高% to 18% of lysine residues, and is a highly basic hydrophilic protein. .

② そのア ミ ノ酸配列中に Ν - グリ コシレーシヨ ン部位は 見いだされない。 .  (2) No Ν-glycosylation site is found in the amino acid sequence. .

③ 10個のシスチン残基が見られ高度の高次構造をとつて いるこ とが推察される。  (3) Ten cystine residues are observed, suggesting that they have a high-order structure.

④ 以上の特徵は ΜΚフ ァ ミ リ 一と報告されている細胞 増殖分化に関係している と言われる蛋白質 ΜΚ - 1 と O S F - 1は非常に類似することを示唆する。 ア ミ ノ酸 配列上では Μ Κ - 1 とマウス 0 S F - 1の間に次のよう な 48.3%の相同性が見られる。 ④ These features suggest that proteins ΜΚ-1 and OSF-1, which are reported to be involved in cell proliferation and differentiation, are very similar. On the amino acid sequence, the following 48.3% homology is observed between Μ Κ-1 and mouse 0 SF-1.

1 50 OSF-1: HSSQQYQQOR KKFAAATIJLL IFILAAVDTA EAGKKEKPEK KVKKSDCGEW 1 50 OSF-1: HSSQQYQQOR KKFAAATIJLL IFILAAVDTA EAGKKEKPEK KVKKSDCGEW

51 100 OSF-1: QWSVCVPTSG DCGLGTREGT TG EC QTM KTQRC IPC W KQFG EC 51 100 OSF-1: QWSVCVPTSG DCGLGTREGT TG EC QTM KTQRC IPC W KQFG EC

* * * * ** *** *** *** ** * * * * ** *** *** *** **

MK-1 ： MGF REGT-CGAQT Q VHC VPCN WK EFGADC MK-1: MGF REGT-CGAQT Q VHC VPCN WK EFGADC

1 32  1 32

101 150 OSF-1: YQFQAWGECD LNTA1KT TG SLKRA1HNAD CQKTVTェ S P CGK1T P PQ 101 150 OSF-1: YQFQAWGECD LNTA1KT TG SLKRA1HNAD CQKTVT S P CGK1T P PQ

* * ** ** * * * * ** * * ** * ** * ** * · * * ** ** * * * * ** * * ** * ** * ** *

MK-1 ： Y FESWGACD GSTGTKARQG TL KARYTAQ CQETェ RVTKP CTSKTKS T MK-1: Y FESWGACD GSTGTKARQG TL KARYTAQ CQET RVTKP CTSKTKS T

33 82  33 82

151 168 151 168

OSF-1: AESKKKK EG KKQEKMLD OSF-1: AESKKKK EG KKQEKMLD

* * *  * * *

MK-1 ： A GKG D  MK-1: A GKG D

83 90  83 90

MK - 1 は、 分泌性の蛋白質 （またはポリペプチ ド） で あることが知られており、 レチノイ ン酸支配下のェンプリ ォニッ クカルシノーマ細胞の分化因子であることが示唆さ れており （友村美根子， 門松健治， 村松喬， 生化学， 1989， Voi.61, Να9, Page 1040 ) 、 O S F - 1 も組織特異性が骨 芽細胞と脳細胞である点を除いて MK - 1 に構造が非常に 似ている。 本発明の蛋白質は O S F 一 1のア ミ ノ酸残基の 全てを含むもの、 又は実質的に O S F - 1 と同一の活性を 有する O S F - 1の一部ア ミ ノ酸残基の置換体を含む。 MK-1 is known to be a secreted protein (or polypeptide), and has been suggested to be a differentiation factor for empironic carcinoma cells under the control of retinoic acid (Mineko Tomomura, Kenji Kadomatsu, Takashi Muramatsu, Biochemistry, 1989, Voi.61, Να9, Page 1040), and OSF-1 are very similar in structure to MK-1 except that their tissue specificities are osteoblasts and brain cells. ing. The protein of the present invention may be a protein containing all of the amino acid residues of OSF-11 or a partially substituted amino acid residue of OSF-1 having substantially the same activity as OSF-1. Including.

本発明の蛋白質は配列 〔 I〕 の部分よりなる蛋白質と し て分泌されることが可能である。 MK - 1 との類似性から みて Π番目の G in (Q) から 84番目の A rg (R) の間で開 裂をうけて分子量 9 0前後の蛋白質と して分泌される可能 性がある。 したがって、 本発明の蛋白質は、 配列 〔 I〕 の 部分よりなる蛋白質及びそれと実質上同一の活性を有する 一部ァ ミ ノ酸残基の置換体を含む。 The protein of the present invention can be secreted as a protein consisting of the sequence [I]. Judging from the similarity with MK-1, it can be secreted as a protein with a molecular weight of around 90 after being cleaved from Gin (Q) at position Π to Arg (R) at position 84. There is. Therefore, the protein of the present invention includes a protein consisting of the part of the sequence [I] and a partial amino acid residue having substantially the same activity as the protein.

O S F - 1蛋白質をコー ドする D N A  O S F-1 Coding protein D N A

本発明による蛋白質をコー ドする D N Aは、 前記配列 The DNA encoding the protein according to the present invention has the sequence

〔 I〕 をコー ドする塩基配列を有する もの、 並びに実質的 に O S F - 1 と同一の活性を有する O S F - 1の部分も し く は O S F - 1の一部ァ ミ ノ酸残基の置換体をコー ドする 塩基配列を有するものである。 Having a nucleotide sequence encoding [I], or a substituting part of an OSF-1 or a partial amino acid residue of the OSF-1 having substantially the same activity as OSF-1 It has a base sequence that encodes

本発明による D N Aの典型的な配列は、 下記の配列 〔Π〕 のものである。  A typical sequence of DNA according to the present invention is the following sequence [Π].

ATG-YZG-XCy-CAU-CAU-TAY-CAG-CAG-CAU-CGT-ZGA-AAA-TTT-GCA-GCT- GCC-TTC-Y G-GCA.-TTV-ATT-TTC-ATZ-YTG-GCA-GCT-GTG-GAY-ACT-GCT- GAU-GCZ-GGG^AAG-AAA-GAG-AAA-CCW-GAA-AAA-AAU-GTG-AAU-AAG-TCT- GAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGY-GTG-CCY-ACC-AGY-GGU- GAC-TGT-GGU-YTG-GGC-ACZ-CGG-GAG-GGC-ACT-CGV-ACT-GGZ-GCY-GAG- TGC-AAU-CAU-ACC - ATG-AAG - ACY-CAG - AGA-TCT - AAG-ATC - CCY - TGC - AAC - TGG - AAG - AAG—CAU-TTT - GGZ - GCX - GAG-TGC - AAU - TAG - CAG - TTC - CAG二 GCY - TGG-GGA-GAA-TGT-GAC-C V-AAy-ACZ-GCC-Y G-AAG-ACC-AGA-ACT-GGZ - AGY-CTG-AAG-CGA-GCY-CTG-CAC-AAT-GCY-GAZ-TGY-CAG-AAU-ACT-GTC- ACC - C - TCC - AAG - CCC - TGT - GGC - AAU-CTV - ACC - AAG - CCC - AAU - CCT - CAA - G U - GAU- TCW - AAG - AAG - AAG - AAA - AAG - GAA- GGC - AAG - AAA - CAG - GAG - AAG- ATG-CTG-GAT-TAA  ATG-YZG-XCy-CAU-CAU-TAY-CAG-CAG-CAU-CGT-ZGA-AAA-TTT-GCA-GCT- GCC-TTC-Y G-GCA.-TTV-ATT-TTC-ATZ-YTG- GCA-GCT-GTG-GAY-ACT-GCT- GAU-GCZ-GGG ^ AAG-AAA-GAG-AAA-CCW-GAA-AAA-AAU-GTG-AAU-AAG-TCT- GAC-TGT-GGA-GAA- TGG-CAG-TGG-AGT-GTG-TGY-GTG-CCY-ACC-AGY-GGU-GAC-TGT-GGU-YTG-GGC-ACZ-CGG-GAG-GGC-ACT-CGV-ACT-GGZ-GCY- GAG- TGC-AAU-CAU-ACC-ATG-AAG-ACY-CAG-AGA-TCT-AAG-ATC-CCY-TGC-AAC-TGG-AAG-AAG-CAU-TTT-GGZ-GCX-GAG-TGC- AAU-TAG-CAG-TTC-CAG2 GCY-TGG-GGA-GAA-TGT-GAC-C V-AAy-ACZ-GCC-Y G-AAG-ACC-AGA-ACT-GGZ-AGY-CTG-AAG- CGA-GCY-CTG-CAC-AAT-GCY-GAZ-TGY-CAG-AAU-ACT-GTC- ACC-C-TCC-AAG-CCC-TGT-GGC-AAU-CTV-ACC-AAG-CCC-AAU- CCT-CAA-GU-GAU- TCW-AAG-AAG-AAG-AAA-AAG-GAA- GGC-AAG-AAA-CAG-GAG-AAG- ATG-CTG-GAT-TAA

C Π 〕  C Π]

配列中、 Uは Aか G、 Yは Cか T、 Xは Gか T、 Zは A力、 C、 Vは Gか C、 Wは Tか Aである。 In the array, U is A or G, Y is C or T, X is G or T, Z is A force, C and V are G or C, and W is T or A.

マ ウ ス O S F - 1 及び ヒ ト O S F - 1 をコー ドする D N Aの具体例は下記の配列 〔Π - I〕 及び 〔Π - Π〕 で 示される。  Specific examples of DNA encoding mouse OSF-1 and human OSF-1 are represented by the following sequences [Π-I] and [Π-Π].

ATG - TCG -！ TCC一 CAG - - CAG - CAG - CAA - CGT - AGA- AAA - ΤΤΤ一 GCA - GCT - ATG-TCG-! TCC-1 CAG--CAG-CAG-CAA-CGT-AGA- AAA-ΤΤΤ 一 GCA-GCT-

GCC-TTC-CTG-GCA-TTG-ATT-TTC-ATC-TTG-GCA-GCT-GTG-GAC-ACT-GCT- GAG-GCC-GGG-AAG-AAA-GAG-AAA-CCT-GAA-AAA-AAG-GTG-AAA-AAG-TCT.- GAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGC-GTG-CCT-ACC-AGC-GGG- GAC-TGT-GGA-TTG-GGC-ACC-CGG-GAG-GGC-ACT-CGC-ACT-GGC-GCC-GAG- TGC -AAA- CAG-ACC-ATG-AAG-ACT -CAG-AGA-TGT-AAG-ATC- CCT-TGC-AAC- TGG-AAG-A2^-CAG-TTT-GGA-GCT-GAG-TGC-AAG-TAC-C^-TTC-CAG-GCT- TGG-GGA-GAA-TGT-GAC-CTC-AAT-ACC-GCC-TTG-AAG-ACC-AGA-ACT-GGC- AGC-CTG-AAG-CGA-GCT-CTG-CAC-AAT-GCT-GAC-0?GT-CAG-AAA-ACT-GTC- ACC-ATC-TCC-AAG-CCC-TGT-GGC-AAG-CTC-ACC-AAG-CCC-AAG-CCT-CAA- GCG - GAG - TCA-AAG - AAG- AAG二 AAA - AAG - GAA - GGC - AAG - AAA-CAG - GAG - AAG - ATG-CTG-GAT-TAA GCC-TTC-CTG-GCA-TTG-ATT-TTC-ATC-TTG-GCA-GCT-GTG-GAC-ACT-GCT-GAG-GCC-GGG-AAG-AAA-GAG-AAA-CCT-GAA-AAA- AAG-GTG-AAA-AAG-TCT.- GAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGC-GTG-CCT-ACC-AGC-GGG- GAC-TGT-GGA-TTG-GGC -ACC-CGG-GAG-GGC-ACT-CGC-ACT-GGC-GCC-GAG- TGC -AAA- CAG-ACC-ATG-AAG-ACT -CAG-AGA-TGT-AAG-ATC- CCT-TGC-AAC -TGG-AAG-A2 ^ -CAG-TTT-GGA-GCT-GAG-TGC-AAG-TAC-C ^ -TTC-CAG-GCT- TGG-GGA-GAA-TGT-GAC-CTC-AAT-ACC-GCC -TTG-AAG-ACC-AGA-ACT-GGC- AGC-CTG-AAG-CGA-GCT-CTG-CAC-AAT-GCT-GAC-0? GT-CAG-AAA-ACT-GTC- ACC-ATC-TCC -AAG-CCC-TGT-GGC-AAG-CTC-ACC-AAG-CCC-AAG-CCT-CAA- GCG-GAG-TCA-AAG-AAG- AAG Double AAA-AAG-GAA-GGC-AAG-AAA-CAG -GAG-AAG-ATG-CTG-GAT-TAA

〔n - I〕 [N-I]

ATG - CAG - GOT - CAA - CAG-TAC - CAG- CAG - CAG - CGT - CGA - AAA - TTT - GCA - GCT -ATG-CAG-GOT-CAA-CAG-TAC-CAG- CAG-CAG-CGT-CGA-AAA-TTT-GCA-GCT-

GCC-TTC-TTG-GCA-TTC-ATT-TTC-ATA-CTG-GCA-GCT-GTG-GAT-ACT-GCT-GCC-TTC-TTG-GCA-TTC-ATT-TTC-ATA-CTG-GCA-GCT-GTG-GAT-ACT-GCT-

GAA-GCA-GGG-AAG-A¾A-GAG-AAA-CCA-GAA-AAA-AAA-GTG-AAG-AAG-TCT-GAA-GCA-GGG-AAG-A¾A-GAG-AAA-CCA-GAA-AAA-AAA-GTG-AAG-AAG-TCT-

GAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGT-GTG-CCC-ACC-AGT-GGA-GAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGT-GTG-CCC-ACC-AGT-GGA-

GAC-TGT-GGG-CTG-GGC-ACA-CGG-GAG-GGC-ACT-CGG-ACT-GGA-GCT-GAG-GAC-TGT-GGG-CTG-GGC-ACA-CGG-GAG-GGC-ACT-CGG-ACT-GGA-GCT-GAG-

TGC-AAG-CAA - ACC-ATG-AAG-ACC-CAG-AGA-TGT-AAG - ATC-CCC - TGC-AAC -TGC-AAG-CAA-ACC-ATG-AAG-ACC-CAG-AGA-TGT-AAG-ATC-CCC-TGC-AAC-

TGG - AAG - AAG - CAA - TTT - GGC - GCG - GAG - TGC - AAA - TAC - CAG - TTC - CAG - GCC -TGG-AAG-AAG-CAA-TTT-GGC-GCG-GAG-TGC-AAA-TAC-CAG-TTC-CAG-GCC-

TGG-GGA-GAA-TGT-GAC-CTG-AAC-ACA-GCC-CTG-AAG-ACC-AGA-ACT-GGA-TGG-GGA-GAA-TGT-GAC-CTG-AAC-ACA-GCC-CTG-AAG-ACC-AGA-ACT-GGA-

AGT-CTG-AAG-CGA-GCC-CTG-CAC-AAT-GCC-GAA-TGC-CAG-AAG-ACT-GTC-AGT-CTG-AAG-CGA-GCC-CTG-CAC-AAT-GCC-GAA-TGC-CAG-AAG-ACT-GTC-

ACC-ATC-TCC-AAG-CCC-TGT-GGC-AAA-CTG-ACC-AAG-CCC-AAA-CCT-CL¾JV-ACC-ATC-TCC-AAG-CCC-TGT-GGC-AAA-CTG-ACC-AAG-CCC-AAA-CCT-CL¾JV-

GCA-GAA-TCT-AAG-AAG-AAG-AAA-AAG-GAA-GGC-AAG-AAA-CAG-GAG-AAG-GCA-GAA-TCT-AAG-AAG-AAG-AAA-AAG-GAA-GGC-AAG-AAA-CAG-GAG-AAG-

ATG-CTG-GAT-TAA ATG-CTG-GAT-TAA

〔 n - n〕 [N-n]

蛋白質のア ミ ノ酸配列が与えられれば、 それをコー ドす る塩基配列はいわゆる遺伝暗号表を参照して容易に定ま り、 また前記したア ミ ノ酸配列をコー ドする種々の塩基配列を 適宜選択するこ とができる。 従って、 本発明による蛋白質 をコー ドする D N Aとは、 その塩基配列が前記配列 〔Π〕 である もの並びにその縮重異性体である。 こ こで、 「縮重 異性体」 とは、 縮重関係にあるコ ドンが使用されている点 以外は塩基配列が同一で同一の蛋白質をコー ドする D N A を意味する ものとする。  Given the amino acid sequence of a protein, the nucleotide sequence encoding it can be easily determined by referring to the so-called genetic code table, and various bases encoding the amino acid sequence described above. The sequence can be appropriately selected. Therefore, DNA encoding the protein according to the present invention includes those having the nucleotide sequence of the above-mentioned sequence [Π] and degenerate isomers thereof. Here, the term "degenerate isomer" shall mean DNA which has the same nucleotide sequence and encodes the same protein except that codons having a degenerate relationship are used.

O S F - 1蛋白質をコー ドする D N Αの取得  Obtain DNA coding for OSF-1 protein

本発明による D N Aは、 その塩基配列が定ま っているこ とから、 その D N Aを取得するひとつの手段は、 核酸の化 学合成の方法に従って製造することである。 Since the base sequence of the DNA according to the present invention has been determined, one means for obtaining the DNA is to convert nucleic acid. It is to produce according to the method of chemical synthesis.

また本発明によるマウス O S F - 1をコー ドする D N A は、 マウス頭蓋冠由来の骨芽細胞 M C 3 T 3 E 1 (児玉等， 1981, J pn. J. Oi a 1 Biol. , Vo .23, Pages 899- 901)細胞 を直径 emのペ ト リ皿 40枚に 1枚あたり 2.2X 105 個に撒 き込み約 9 日間 °Cで適宜培地を交換し培養後 8 X 1G7 個 になったところで Maniat is等の方法により （Maniatis等， 1982, Mol ecu l ar cloning: A La oratory Manual, Cold Spring Harbor Laboratory) 総 R N Aを調製後通常の方法 により m R N Aの精製、 c D N Aの合成、 ク ローン化によ り製造する こ と もできる。 Further, the DNA encoding mouse OSF-1 according to the present invention can be obtained from a mouse calvarial osteoblast MC3T3E1 (Kodama et al., 1981, Jpn. J. Oia 1 Biol., Vo.23, (Pages 899-901) Cells were seeded in 2.2 × 10 5 cells / plate in 40 Petri dishes with em diameter and the medium was changed appropriately at 9 ° C for about 9 days.After culturing, the cells became 8 × 1G 7 cells. By the way, Maniatis et al. (Maniatis et al., 1982, Molecular Cloning: A Laoratory Manual, Cold Spring Harbor Laboratory) After preparing total RNA, purify mRNA, synthesize cDNA, clone It can also be manufactured by conversion.

またこのマウス由来の c D N Aをプロ一プと してヒ トゃ 他の動物の c D N Aライブラ リ ーから製造することもでき  The cDNA derived from this mouse can also be produced as a probe from the cDNA library of another animal.

0 S F - 1蛋白質の発現 Expression of 0 S F-1 protein

O S F - 1蛋白質の生産は、 宿主は大腸菌、 酵母、 動物 細胞を用い、 それぞれに対応したプロモーターにより行う こ とができる。 その具体的方法は Maniat is等 （前出） 等に 記載の方法を用いることができる。  The OSF-1 protein can be produced using Escherichia coli, yeast, or animal cells as the host, using the corresponding promoter. As a specific method, the method described in Maniatis et al. (Supra) can be used.

以下に実施例により本発明を説明する。 実施例で用いた 手法は次のとおりである。  Hereinafter, the present invention will be described by way of examples. The method used in the examples is as follows.

制限酵素は米国ニュ一 ' イ ングラ ン ド ' バイオラブス社 (New England Biolabs) 、 及び宝酒造から入手し、 そして 特にことわらない限り製造者の指示に従って使用した。 キ 口 シークェンス用デレーン ヨ ンキッ ト、 ク ローニングべク 3 ター P U C 118, T 4 D N A リ ガーゼ及び、 細菌性アル力 リ性ホスファタ一ゼは、 宝酒造から入手し、 製造者の指示 に従って使用した。 c D N A合成システム · プラスはアマ シャム社， イギリ ス， （Amersham) から入手し製造者の指 示に従って使用した。 A MV リバース ト ラ ンスク リ プター ゼ、 m R N A精製キ ッ ト及び、 E c Q R I - N o t I ァダ プタ一はフ アルマシア社， スウェーデン， （Pharmacia) か ら入手し製造者の指示に従って使用した。 ラ ンダムプライ ム ド D N Aラベ リ ングキッ ト はべー リ ンガーマ ンハイ ム 山之内より入手し製造者の指示に従って使用した。 i gtio ク ロ一ニングベク タ一と、 ィ ンビ ト ロノ、0ッ ケージングキッ ト 「ギガパッ ク ゴール ド （G i g a p a c k Gold) 」 は、 ス トラタ ジーン社， 米国， （Stratagene) より入手し製造者の指示 に従って使用した。 大腸菌（E. coli) の形質転換、 D NA のライゲーシヨ ンは Mania tis等 （前掲） により記載された 方法により行った。 Restriction enzymes were obtained from New England Biolabs, USA, and Takara Shuzo, and were used according to the manufacturer's instructions unless otherwise noted. Exit Delane kit for sequence, cloning vector 3-ter PUC 118, T4 DNA ligase and bacterial phosphatase were obtained from Takara Shuzo and used according to the manufacturer's instructions. c DNA synthesis system · Plus was obtained from Amersham, UK, and used according to the manufacturer's instructions. AMV reverse transcriptase, mRNA purification kit, and Ec QRI-Not I adapter were obtained from Pharmacia, Sweden, and used according to the manufacturer's instructions. . Random primed DNA labeling kits were obtained from Behringer Mannheim Yamanouchi and used according to the manufacturer's instructions. i gtio cloning vector and invitrono, zero packaging kit "Gigapack Gold" were obtained from Stratagene, USA, (Stratagene) and the manufacturer's instructions. Used according to. Transformation of Escherichia coli (E. coli) and ligation of DNA were performed according to the method described by Mania tis et al.

図面の簡単な説明 BRIEF DESCRIPTION OF THE FIGURES

第 1図は、 プラ ス ミ ド p M C 031の構造を示す。  FIG. 1 shows the structure of the plasmid pMC031.

白帯はマウス O S F ― 1の c D N A挿入部を示す。 矢印 はアンピシリ ン耐性遺伝子を示す。 黒丸はプラス ミ ドの複 製開始点を示す。  The white band indicates the cDNA insertion site of mouse OSF-1. The arrow indicates the ampicillin resistance gene. The black circles indicate the starting point of plasmid replication.

第 2図は、 プラス ミ ド P M C G31 及び、 10個のデレー シヨ ン ミ ュータ ン トの c D N A挿入部の構造及び、 塩基配 列を決定した領域を示す。  FIG. 2 shows the structure of the plasmid PMCG31 and the cDNA inserts of the 10 deletion mutants, and the region in which the base sequence was determined.

実線は、 各プラス ミ ドに含まれる c D N A挿入部を示す。 矢印は、 塩基配列を決定した領域及び方向を示す。 The solid line indicates the cDNA insert contained in each plasmid. Arrows indicate the region and direction in which the base sequence was determined.

第 3図は、 p M C l の c D N A挿入部の全塩基配列及 び、 塩基配列から推定されるマウス 0 S F - 1のア ミ ノ酸 配列を示す。  FIG. 3 shows the entire nucleotide sequence of the cDNA insertion site of pMCl and the amino acid sequence of mouse 0 S F-1 deduced from the nucleotide sequence.

下線は E coR I - Not Iアダプターに由来する部分を示 す。 * * *は転写終止コ ドンを示す。  The underline indicates the portion derived from the EcoRI-NotI adapter. *** indicates a transcription termination codon.

第 4図は、 ノ ーザンプロ ッティ ングによる、 0 S F - 1 遺伝子の組織特異的発現の検討を示す。  FIG. 4 shows examination of tissue-specific expression of the 0SF-1 gene by Northern plotting.

各 レー ン は次の通 り 。 1 ； 胸腺、 2 ； 脾臓、 3 ； 脳、 4 ; 腎臓、 5 ; 肝臓、 6 ; 肺、 7 ; 骨格筋、 8 ; Each lane is as follows. 1; thymus, 2; spleen, 3; brain, 4; kidney, 5; liver, 6; lung, 7; skeletal muscle, 8;

MC 3 T 3 E 1細胞、 9 ; N I H 3 T 3細胞。 28S， 18 S はリ ポソ一ム RN Aの位置を示す。 MC3T3E1 cells, 9; NIH3T3 cells. 28S and 18S indicate the position of the liposome RNA.

第 5図は、 プラス ミ ド P H S G757 の構造を示す。  FIG. 5 shows the structure of plasmid PHSG757.

白帯はポリ Aシグナル、 矢印付白帯は S V 40T抗原初期 プロモーター、 矢印はク ロラムフヱニコール耐性遺伝子を 示す。 黒丸はプラス ミ ドの複製開始点を示す。  The white band indicates the poly A signal, the white band with the arrow indicates the SV40T antigen early promoter, and the arrow indicates the chloramphenicol resistance gene. The black circle indicates the origin of plasmid replication.

第 6図は、 p H B R 1の c D Ν Α揷入部の全塩基配列及 び、 塩基配列から推定される ヒ ト O S F - 1のア ミ ノ酸配 列を示す。  FIG. 6 shows the entire nucleotide sequence of the cDNA fragment of pHBR1 and the amino acid sequence of human OSF-1 deduced from the nucleotide sequence.

発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION

次に実施例を示して本発明をさ らに具体的に説明する。 実 施 例 1 マ ウ ス頭蓋冠由来骨芽細胞様細胞株  Next, the present invention will be described more specifically with reference to examples. Example 1 Mouse calvaria-derived osteoblast-like cell line

M C 3 T 3 E 1細胞由来 c D N Aライ ブラ リ 一の作製  Preparation of cDNA library derived from MC3T3E1 cells

約 8 X 107 個のマウス骨芽細胞様細胞株 M C 3 T 3 E 1 一 1.5— 細胞 （児玉等， 1981, Jpn. J. Ora l Biol. , 23, 899 - 901 ) より、 グァニジン法 （Mani at i s等， 前掲） により総 R N A を調製後、 フ ア ルマ シ ア の m R N A精製キ ッ ト を用い m R N Aを調製した。 調製した m R N Aを铸型と して、 c D N A合成システム · プラ ス （アマ シャ ム社） によ り 、About 8 x 10 7 mouse osteoblast-like cell lines MC 3 T 3 E 1 I. Total RNA was prepared from 1.5-cells (Kodama et al., 1981, Jpn. J. Oral Biol., 23, 899-901) by the guanidine method (Mani at is, et al., Supra), and then the total RNA was prepared. mRNA was prepared using an mRNA purification kit. Using the prepared mRNA as type I, a cDNA synthesis system plus (Amersham)

2本鎖 c D N Aを合成した。 合成された 2本鎖 c D N Aに まず E c 0 R I - N 01 I アダプタ一 （フ ア ルマ シア社） を T 4 D N A リ ガーゼを用いて連結し、 次に E co R I で消化 した； I gtlOク ローニ ングベクター （ス トラタジー ン社） を、 T 4 D N A リ ガ一ゼを用いて連結した。 ；i gtlOと連結した c D N Aを、 ス ト ラ 夕 ジー ンのイ ン ビ ト ロノヽ0ッ ケー ジ ング キッ ト 「ギガパッ クゴール ド（G i g a p a c It Gold) 」 を用いて イ ン ビ ト ロ （in v i t ro) でラ ムダフ ァ ー ジにノ、° ッ ケー ジ し、 S M緩衝液 （Mani at i s等， 前掲） 中に保存した。 これ を大腸菌 C 600 株 （国立予防衛生研究所 遺伝子バ ン クDouble-stranded cDNA was synthesized. First, Ec0RI-N01I adapter-1 (Pharmacia) was ligated to the synthesized double-stranded cDNA using T4 DNA ligase, and then digested with EcoRI; Cloning vectors (Stratagene) were ligated using T4 DNA ligase. ; The i gtlO c DNA was linked to a, b down bi-DOO be sampled La evening Gee down Russia Nono 0 Tsu cage packaging kit "Gigapa' Kugoru de (G igapac It Gold)" using a stomach down bi collected by filtration ( The cells were packaged in a lambda phage in vitro and stored in SM buffer (Mani at is, et al., supra). This was transformed into E. coli C600 strain (National Institute of Preventive Health Gene Bank).

H T 003 )に感染させたところ、 1 gの c D N A当たり、 約 1. 4 X 107 個の一次フ ァージプラークの頻度で c D N Aラ イブラ リ 一が作成された。 HT 003), a cDNA library was generated at a frequency of approximately 1.4 × 10 7 primary phage plaques per gram of cDNA.

実 施 例 2 M C 3 T 3 E 1細胞由来 c D N Aライブラ リ ーの、 N I H 3 T 3細胞（ATCC CRL 1658 ) と の ディ フ ァ レ ン シ ヤ ノレス ク リ ーニ ン グ d i f f e r en t i a l s c r e en i n ) に よ る 、 M C 3 T 3 E 1細胞特異的ク ロー ンの選択 及び、 プラス ミ ドベクター p U C l U への ク ロ ーニ ング M C 3 T 3 E 1細胞自身の mR N A及び、 M C 3 T 3 EExample 2 Difference library of cDNA library derived from MC3T3E1 cells with NIH3T3 cells (ATCC CRL 1658). ) To select clones specific to MC3T3E1 cells and to clone the plasmid vector pUCIU. MC 3 T 3 E 1 cells own mRNA and MC 3 T 3 E

1 細胞 と 同様の方法で調製 したマ ウ ス繊維芽細胞株 N I H 3 T 3細胞（ATCC CRL 1658) の mR N Aを铸型と し て、 AMVリ ノく一ス トラ ンスク リプターゼ （フアルマシア） によりそれぞれ放射性 c D N Aプローブを合成した。 前記 の M C 3 T 3 E 1細胞由来の c D N Aライブラ リ ーのうち、 約 1. 5 X 104 個を、 それぞれの放射性 c D N Aプローブを 用いて、 プラークハイプリ ダイゼーシヨ ン法 （Mania t is等， 前掲） によって、 順次スク リ ーニングし、 M C 3 T 3 E 1 細胞由来の放射性 c D N Aプローブに特異的にハイプリ ダ ィズする 78個のクローンを得た。 Using the mRNA of mouse fibroblast cell line NIH 3T3 cells (ATCC CRL 1658) prepared in the same manner as 1 cells as type III, the cells were treated with AMV Linokin Strand Scryptase (Pharmacia), respectively. A radioactive cDNA probe was synthesized. Of the above-mentioned cDNA library derived from MC3T3E1 cells, about 1.5 × 10 4 cells were subjected to the plaque-hybridization method (Maniatis, et al.) Using each radioactive cDNA probe. According to the above, screening was performed sequentially to obtain 78 clones specifically hybridizing to a radioactive cDNA probe derived from MC3T3E1 cells.

次に、 これらのファージク ローンの溶菌物からそれぞれ のファージ D N Aをフヱノール処理に続く ェタノ ール沈殿 によ り精製し （Maniat i s等； 前掲） 、 E coR I で消化、 ァガロースゲル電気泳動により分離後、 c D N A挿入部の みを精製した （Maniatis等， 前掲） 。 これらの c D N A揷 入部を基質と して、 ランダムプライム ド D N Aラベリ ング キッ ト （ベ一リ ンガーマンハイム山之内） を用いてそれぞ れのク ローン由来の放射性プローブを調製した。  Next, each phage DNA was purified from the lysate of these phage clones by phenol treatment followed by ethanol precipitation (Maniat is et al .; supra), digested with EcoRI, separated by agarose gel electrophoresis, c Only the DNA insert was purified (Maniatis et al., supra). Using these cDNA insertion sites as substrates, radioactive probes derived from each clone were prepared using a random primed DNA labeling kit (Berlingermanheim Yamanouchi).

他方、 M C 3 T 3 E 1細胞及び、 N I H 3 T 3細胞の m R N A 0. 3 ^を、 ホルムアルデヒ ド変性ァガロース ゲル電気泳動 （Mani at i s等， 前掲） により分離後、 ナイ ロ ン膜 （BYODYNE, Pa l l Bio Suppor t, 米国） に固定した ( aniatis^, 前掲） 0 On the other hand, the mRNA 0.3 ^ of MC3T3E1 cells and NIH3T3 cells was separated by formaldehyde denaturing agarose gel electrophoresis (Mani at is, et al., Supra), and then separated by a nylon membrane (BYODYNE , Pall Bio Support, USA) (aniatis ^, supra) 0

個 々 の ク ロ ー ン由来の放射性プロ ー ブを用いて、 ノ ー ザ ン プ ロ ッ テ ィ ン グ に よ っ て、 各 ク ロ ー ン の M C 3 T 3 E 1細胞及び、 N I H 3 T 3細胞での発現量を 定量し、 M C 3 T 3 E 1細胞に特異的に発現している 1個 のク ローンを得た。 このク ローンを M C 031 と称する。 Using radioactive probes from individual clones, By Northern blotting, the expression level of each clone in MC3T3E1 cells and NIH3T3 cells was quantified, and MC3T3E1 cells were quantified. One clone specifically expressed in cells was obtained. This clone is referred to as MC031.

このフ ァージク ローン M C 031 の c D N A挿入部を、 制 限酵素 E co R I で消化し 5 ' 末端のリ ン酸を細菌性アル力 リ性ホスフ ァターゼ （宝酒造） 処理により除去したク ロー ニングベク タ一 P U C 118 (宝酒造） と、 T 4 D N A リ ガーゼを用いて連結しプラス ミ ド p M C l を得た。 その 構造を第 1図に示す。  The cDNA insert of this Phage clone MC031 was digested with the restriction enzyme EcoRI, and the 5'-terminal phosphoric acid was removed by treatment with bacterial alkaline phosphatase (Takara Shuzo). It was ligated with PUC118 (Takara Shuzo) using T4 DNA ligase to obtain plasmid pMCl. Fig. 1 shows the structure.

実 施 例 3 p M C 031 の c D N A挿入部の塩基配列の 決定  Example 3 Determination of the nucleotide sequence of the cDNA insertion site of pMC031

p M C 031 を制限酵素 S ph I及び、 B amH I で消化後、 キロシークェンス用デレージヨ ンキッ ト （宝酒造） によつ て 10個のデレ一シヨ ン ミ ュータ ン トを作成した。 これらを、 p M C 031- 1 , p M C 031-2 , p M C 031- 3 , ·■·, p M C 03卜 10と称する （第 2図） 。 アプライ ド ' バイオシステム ズ社， 米国， （App l i ed B i osys t ems) の自動塩基配列解析 装置 （Mode l 370 A) を用いて、 p M C 031 及びこれらのデ レーシ ヨ ン ミ ュータ ン トの H ind mサイ ト側よ り c D N A 挿入部の塩基配列、 それぞれ約 300 塩基対を決定した。 そ の結果得られた c D N A挿入部の全塩基配列及び、 それか ら推定されるア ミ ノ酸配列を第 3図に示す。 また、 この遺 伝子によ ってコ ー ドされる と推定される蛋白質をマウス O S F - 1 と称する。 実 施 例 4 マウス O S F - 1の組織特異的発現の検討 マウス 0 S F - 1 の遺伝子の組織特異的発現をノ 一ザン プロ ッティ ング法によって検討した。 After digesting pMC031 with the restriction enzymes SphI and BamHI, ten deletion mitants were prepared using a kilosequence deletion kit (Takara Shuzo). These are referred to as pMC031-1, pMC031-2, pMC031-3,..., PMC0310 (FIG. 2). Applied Biosystems, Inc., USA, (Applied Biosystems), using an automatic sequence analyzer (Model 370A), pMC031, and their displacement mutators. Approximately 300 base pairs were determined from the Hind site side of the cDNA insertion site. FIG. 3 shows the entire nucleotide sequence of the resulting cDNA insert and the amino acid sequence deduced therefrom. The protein presumed to be coded by this gene is called mouse OSF-1. Example 4 Examination of Tissue-Specific Expression of Mouse OSF-1 Tissue-specific expression of mouse 0SF-1 gene was examined by Northern blotting.

ク レア社よ り 購入 した 4週令のマウ ス （ C 57 B L Z 6 N) 10匹の胸腺、 脾臓、 脳、 腎臓、 肺、 肝臓、 骨格筋 か ら、 グァニ ジ ン法 （ M a n i a t i s等， 前掲） によ っ て それぞれの総 R N Aを調製した。 これらの総 R N A及び、 M C 3 T 3 E 1細胞、 N I H 3 T 3細胞より調製した総 R N Aそれぞれ 1 ϋ i«gを、 ホルムアルデヒ ド変性ァガロー スゲル電気泳動により分離後、 ナイ ロ ン膜（BYODYNE, Pal 1 Bio Suppor t)に固定した （Maniatis等， 前掲） 。  Mouse (C57BLZ6N), 4 weeks old, purchased from Clair, Inc. From 10 thymus, spleen, brain, kidney, lung, liver, and skeletal muscle, guanidine method (Maniatis et al., Supra) ) Was used to prepare each total RNA. After separating these total RNAs and total RNA prepared from MC3T3E1 cells and NIH3T3 cells by 1 μi «g, respectively, by formaldehyde denaturing agarose gel electrophoresis, a nylon membrane (BYODYNE, Pal 1 Bio Support (Maniatis et al., Supra).

他方、 p M C l の c D N A挿入部を E coR I で消化し た後、 ァガロースゲル電気泳動によって精製し （Maniatis 等， 前掲） 、 これを基質と じてべ一リ ンガーマンハイム山 之内のラ ンダムプライム ド D N Aラベリ ングキッ トを用い て放射性プローブを調製した。  On the other hand, the cDNA insert of pMCl was digested with EcoRI and purified by agarose gel electrophoresis (Maniatis et al., Supra), and this was used as a substrate for randomization in the Behringermannheim Yamanouchi. Radioactive probes were prepared using the primed DNA labeling kit.

これらのナイ 口 ン膜とプローブを用いてノ一ザンブロ ッ ティ ングを行った結果、 M C 3 T 3 E 1細胞に強い発現、 脳及び N I H 3 T 3細胞に弱い発現がみられた （第 4図） , 実 施 例 5 ヒ ト O S F - 1遺伝子のク ローニング  As a result of Northern blotting using these nylon membranes and the probe, strong expression was observed in MC3T3E1 cells, and weak expression was observed in brain and NIH3T3 cells (No. 4). Fig.), Example 5 Cloning of human OSF-1 gene

マウス 0 S F - 1遺伝子を有する p M C 031をプローブ と してヒ ト大脳上側頭回 c D N Aライブラ リ ー （クローン テッ ク社， C L H L 10 a) 4 x ld4 ク ロー ンをプラークハ イブリ ダィゼ一シヨ ン法 （Mani a t i s等， 前掲） により選択 し、 1 個の フ ァ ー ジ ク ロ ー ンを得た。 こ の フ ァ ー ジ ク ロ ー ンを H B R l と称する。 さ らに、 こ の フ ァ ー ジク ローン H B R 1の c D N A挿入部を、 制限酵素 E coR I で 消化 し ク ロ ーニ ングベク タ 一 P U C 118 (宝酒造） と、 T 4 D N A リ ガ一ゼを用いて連結しプラス ミ ド p M C 031 と類似の p H B R 1を得た。 Mouse 0 cDNA library with pMC031 containing SF-1 gene as a probe cDNA library of human cerebral upper temporal gyrus (Clontech, CLHL 10a) 4 x ld 4 clones were plaque hybridized One phage clone was obtained according to the method of choice (Mani atis et al., Supra). This fuzzy The loan is called HBR l. In addition, the cDNA insert of this phage clone HBR1 is digested with the restriction enzyme EcoRI and cloned with PUC 118 (Takara Shuzo) and T4 DNA ligase. And ligated to obtain pHBR1 similar to plasmid pMC031.

実 施 例 6 p H B R lの c D N A挿入部の塩基配列の 決定  Example 6 Determination of the nucleotide sequence of the cDNA insertion site of pHBR1

p H B R 1を制限酵素 P s t I及び、 B amH I で消化後、 キロシークェンス用デレーシ ヨ ンキッ ト （宝酒造， 前掲） によって 10個のデレージヨ ンミ ュータ ン トを得た。 これら を p H B R - 1， p H B R 1 - 2 , p H B R l - 3， ……， p H B R 1 - 10と称した。 D N Aシークェンシングキッ ト ( S e q u e n a s e Vers i on 2. 0, Uni ted States Biochemi ca l Co r p o r a t o n) を用いて、 p B R 1及びこれらのデレー シ ヨ ン ミ ュータ ン ト H ind mサイ ト側より c D N A揷入部 の塩基配列、 それぞれ約 200塩基対を決定した。 その結果 得られた c D N A挿入部の全塩基配列及び、 それから推定 されるア ミ ノ酸配列を第 6図に示す。  After digesting pHBR1 with the restriction enzymes PstI and BamHI, ten deletion mitants were obtained using a desorption kit for kilo-sequence (Takara Shuzo, supra). These were named pHBR-1, pHBR1-2, pHBRl-3, ..., pHBR1-10. Using a DNA sequencing kit (Sequenase Version 2.0, United States Biochemi- cal Corporaton), from pBR1 and their departure mitigation Hind m site Approximately 200 base pairs were determined for each of the base sequences of the cDNA insertion part. FIG. 6 shows the entire nucleotide sequence of the resulting cDNA insert and the amino acid sequence deduced therefrom.

実 施 例 7 0 S F - 1蛋白質を発現する発現ベク ター 及びそのべクタ一を使った同蛋白質の生産Example 7 Expression Vector Expressing 0 SF-1 Protein and Production of the Protein Using the Vector

0 S F - 1 の生産は、 特開昭 63 - 7288に記述されてい る増幅された遺伝子によって動物細胞で効率良く 蛋白質を 生産する方法を用いて行った。 〇 S F - 1をこの方法で発 現させるために、 同蛋白質をコ一 ドする c D N Aをカセ ッ トベク ター p H S G (国立予防衛生研究所 遺伝子バン ク登録番号 V E H 7)の S V40ウィルス T抗原初期プロモ一 ターと、 ポリ Αシグナルの間に、 以下のようにして挿入し た。 p H S G747 の S V 40T抗原初期プロモーター上流の E coR I部位を、 E coR I による消化、 末端の Kl enowフラ グメ ン ト （宝酒造） による平滑化、 及びそれに引続く リ ガーゼによる再結合により消失させた。 さ らに、 S V40T 抗原初期プロモータ一とポリ Aシグナル配列の間にある単 — P st l部位を合成 E coR I リ ンカーオリ ゴヌ ク レオチ ド (宝酒造） で置き換え、 同位置に単一 E coR I部位を持つ. イ ンビ ト ロ遺伝子増幅用のプラス ミ ドベク ター p H S G 757 を構築した （第 5図） 。 この E coR I部位に E coR I 部位で挟まれた O S F - 1 c D NA断片を正方向に挿入し プラス ミ ド p O F lを得た。 このプラス ミ ドを制限酵素 B s t X Iで消化することにより、 以下のような両端に非対 称性粘着性末端を持つた 0 S F - 1の発現単位 D N A断片 を得ることができる。 0 SF-1 was produced using the method described in JP-A-63-7288 for efficiently producing a protein in animal cells using the amplified gene.た め In order to express SF-1 by this method, cDNA encoding the same protein must be transferred to cassette vector p HSG (National Institute of Health). Insertion number VEH 7) was inserted between the SV40 virus T antigen early promoter and the polymerase II signal as follows. The EcoRI site upstream of the SV40T antigen early promoter of pHSG747 was eliminated by digestion with EcoRI, blunting of the terminal with the Klenow fragment (Takara Shuzo), and subsequent recombination with ligase. Was. In addition, the single Pst1 site between the SV40T antigen early promoter and the polyA signal sequence was replaced with a synthetic EcoRI linker (oligonucleotide) (Takara Shuzo), and a single EcoR at the same position. It has an I site. A plasmid pHSG757 was constructed for amplification of the in vitro gene (Fig. 5). An OSF-1 cDNA fragment sandwiched between the EcoRI sites was inserted in the EcoRI site in the forward direction to obtain plasmid pOFl. By digesting this plasmid with the restriction enzyme BstXI, an expression unit DNA fragment of 0SF-1 having asymmetric sticky ends at both ends as described below can be obtained.

5' CTGG 0 S F - 1発現 CCACGGGG 3' 3' CCCCGACC 単位 D N A断片 GGTG 5' 5 'CTGG 0 S F-1 expression CCACGGGG 3' 3 'CCCCGACC unit DNA fragment GGTG 5'

Cm) しかし、 p 0 F 1を直ちに B s t X Iで消化したのでは、 同 プラス ミ ドの他の B stX I消化断片と分子量が近似してお り同断片が混在して分離精製が困難なため、 ポリ Aシグナ ル配列下流の単一 X ho I部位にプラス ミ ド p H S G ll (国 立予防衛生研究所 遺伝子バンク 登録番号 V E 9)を挿 入し、 プラス ミ ド p O F 3を構築した。 However, if p0F1 was immediately digested with BstXI, the molecular weight was similar to that of other BstXI digested fragments of the same plasmid, and the same fragments were mixed, making separation and purification difficult. For this purpose, plasmid pHSGII (National Institute of Health and Prevention Genebank registration number VE9) was inserted into a single XhoI site downstream of the polyA signal sequence. To construct plasmid pOF3.

このようにして得た 〔m〕 の断片を、 コス ミ ドベクタ一 p H S G 293 (国立予防衛生研究所 遺伝子バンク 登録番 号 V E 046)の D N Aより B s t X I消化により調整した下記 の D N A断片 〔IV〕 と 20 : 1の分子比で混合し上記特開昭 に記述されている方法により T 4 リガーゼによりタ ンデム に連結した。  The fragment of [m] obtained in this manner was prepared by digesting the DNA of Cosmid Vector-p HSG 293 (National Institute of Preventive Health, Gene Bank registration number VE 046) with BstXI digestion as follows: And 20: 1, and ligated to tandem by T4 ligase according to the method described in the above-mentioned JP-A-sho.

5' CTGG 不ォ ナ + c 0 s CCACGGGG 3' 3' CCCCGACC サイ ト D N A断片 GGTG 5' 5 'CTGG Non + + 0 s CCACGGGG 3' 3 'CCCCGACC Site DNA fragment GGTG 5'

〔IV〕 こ う して得られた長鎖の D N A断片を記述の方法により (Takeshi ta e t a I, 1988, Gene, Vo^.71, Pages 9— 18) 、 ラムダファ一ジ粒子にパッケージし大腸菌 O m206 (工業技 術院微生物工業技術研究所 微ェ研菌寄第 9110号 （FERM P- 9110) )細胞内で増幅後ラムダフ ァ ー ジ粒子内に細胞内 パッケージを行いコス ミ ド D N Aと して大量に調整した。 この D N Aを常法によ り リ ン酸カルシウム共沈法によ り チャ イニーズハムスター卵巣細胞株 （ C H O株） に導入 し、 G 8 耐性のク ロー ンを選択した。 さ らに得られた各 ク ローンから Man i a t i s等 （前掲） の方法により総 R N Aを 調整し O S F - 1 c D N A断片をプローブにしノ ーザンブ ロ ッテイ ング法 （Man i a ί i s等， 前掲） によ り同遺伝子を高 い効率で発現する株を得た。 この選択した O S F - 1産生 株を 10%の牛胎児血清を含む - M E M培地 （G I B C 0 ) で 時間培養し、 その培養上清中に、 O S F - 1を検出し た。 [IV] The long-chain DNA fragment thus obtained was packaged into lambda phage particles by the method described (Takeshita eta I, 1988, Gene, Vo ^ .71, Pages 9-18), and m206 (FERM P-9110) Microbial Industry Research Institute, Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology (AMP) After amplification in cells, package them in lambda phage particles and package them as cosmid DNA. Adjusted a lot. This DNA was introduced into a Chinese hamster ovary cell line (CHO strain) by a conventional method using calcium phosphate coprecipitation, and G8-resistant clones were selected. From each of the obtained clones, total RNA was prepared by the method of Maniatis et al. (Supra) and the OSF-1 cDNA fragment was used as a probe by the northern blotting method (Maniais et al., Supra). A strain that expressed the same gene with high efficiency was obtained. The selected OSF-1 producing strain contains 10% fetal bovine serum-MEM medium (GIBC 0) And OSF-1 was detected in the culture supernatant.

産業上の利用可能性 Industrial applicability

本発明によれば、 骨細胞分化形成能を有する新規な蛋白 質、 それをコー ドする D N Aおよびその遺伝子工学的製法 によるこの蛋白質の製造方法が提供される。 本発明によつ て提供される新規な蛋白質は老人性の疾患と しての骨粗 ショ ゥ症および痴呆症の治療に利用される可能性を有して いる。  According to the present invention, there is provided a novel protein having osteocyte differentiation-forming ability, DNA encoding the same, and a method for producing the protein by a genetic engineering method. The novel protein provided by the present invention has the potential to be used for the treatment of osteoporosis and dementia as senile diseases.

Claims

請 求 の 範 囲 The scope of the claims 1) 下記の配列 〔 I〕 で表されるァ ミ ノ酸配列の全体、 そ の部分的に修飾されたア ミ ノ酸配列の全体またはそれら の部分を有する蛋白質。 1) A protein having the entire amino acid sequence represented by the following sequence [I], the entire amino acid sequence partially modified, or a portion thereof. Met— A*2 - Gin— Gin - Tyr— Gin - Gin— Gin - A g— Arg-Lys - Phe-Ala— Ala— Ala- Phe- Leu - Ala - A*3— lie - Phe -！: le - Leu— Ala-Ala- Val - Asp-Thr-Ala - Glu - Ala - Glv - Lys - I vs - Glu— Lys— Pro - Glu - Lys— Lys - Val - Lys - Lys— Ser - Asp-Cys-Gly-Glu-Trp-Gln-Trp-Ser-Val-Cys-Val-Pro-Thr-Ser-Gly- Asp - Cys - Gly - Leu - Gly- Thr- Arg - Glu— Gly - Thxr—Arg - Thr— Gly - Ala - Glii - Cys - IiVS— Gin - Thir— Met - Iiys -! Thr— Gin - Arg— Cys— Lys -ェ le - Pro— Cys - Asn— Trp - Lys - Lys - Gin - Phe— Gly - Ala - Glu— Cys— Lys - Tyr - Gin— Phe - Gin - Ala - T p - Gly - Glu - Cys -Asp-Leu-Asn-Thr-Ala-I^ -Lys-Thr-Axg-Thr-Gly- Ser-Leu-Lys-Arg-A a-I^u-His-Asn^Ala-A*4-Cys-Gln-Lys-Thr-Val- Thr -ェ le - Ser— Lys - Pro - Cys— Gly - Lys Leu— Thr - Lys - Pro— Lys - Pro— Gin— Ala"Glu-Ser-Lys-Lys-Lys-Lys-Lvs--Glu-Gly--Ly3-Ijys-Gln-Glu-Lys- Met-Leu-Asp Met—A * 2—Gin—Gin—Tyr—Gin—Gin—Gin—Ag—Arg-Lys—Phe-Ala—Ala—Ala-Phe-Leu—Ala—A * 3—lie—Phe—! : le-Leu— Ala-Ala- Val-Asp-Thr-Ala-Glu-Ala-Glv-Lys-I vs-Glu— Lys— Pro-Glu-Lys— Lys-Val-Lys-Lys— Ser-Asp- Cys-Gly-Glu-Trp-Gln-Trp-Ser-Val-Cys-Val-Pro-Thr-Ser-Gly-Asp-Cys-Gly-Leu-Gly- Thr-Arg-Glu— Gly-Thxr—Arg- Thr— Gly-Ala-Glii-Cys-IiVS— Gin-Thir— Met-Iiys-! Thr— Gin-Arg— Cys— Lys-Cell-Pro— Cys-Asn— Trp-Lys-Lys-Gin-Phe— Gly-Ala-Glu— Cys— Lys-Tyr-Gin— Phe-Gin-Ala-Tp-Gly-Glu-Cys -Asp-Leu-Asn-Thr-Ala-I ^ -Lys-Thr-Axg-Thr- Gly- Ser-Leu-Lys-Arg-A aI ^ u-His-Asn ^ Ala-A * 4-Cys-Gln-Lys-Thr-Val- Thr-cell- Ser— Lys-Pro-Cys— Gly- Lys Leu— Thr-Lys-Pro— Lys-Pro— Gin— Ala "Glu-Ser-Lys-Lys-Lys-Lys-Lvs--Glu-Gly--Ly3-Ijys-Gln-Glu-Lys- Met-Leu -Asp 〔 I〕 配列中、 A * 1 は S e rか G I n、 A * 2 は S e rか A 1 a、 A * 3 は L e uか P h e、 A* 4 は A s pか G 1 u、 である。  [I] In the sequence, A * 1 is Ser or GIn, A * 2 is Ser or A1a, A * 3 is Leu or Phe, A * 4 is Asp or G1u. . 2) 下記の配列 〔 I 一 I〕 で表される請求項 1記載の蛋白 M t-Ser-Ser-Gln-Gln-Tyr-Gln-Gln-G n-Arg-Arg-Lys-Phe-Ala-Ala- Ala— Phe - Leu— Ala - Leu -ェ le - Phe—ェ le - Leu— Ala— Ala - Val - Asp - Thi: - Ala- Gl -Ala-Gly-Lys-Lys-Glu-Lys-Pro-Glu-Lys-Lys-Val-Lys-Lys-Ser- Asp-Cys-Gly-Gl -Trp-Gln-Trp-Ser-Val-Cys-Val-Pro-Thr-Ser-Gly- Asp-Cys-Gly-Le -Gly-Thr-Arg-Glu-Gly-Thr-Arg-Thr-Gly-Ala-Glu- Cys— Iiys - Gin - Th - Met— Lys - Thi: - Gin— Arg - Cvs - Lys—ェ le - Pro - Cys— Asii- Trp - Lys - Lys - Gin— Phe - Gly - Ala - Glu - Cys - Iiys - Tyr - Gin— Phe - Gin— Ala- Trp - Glv - Glu - Cys— Asp— Leu - Asn - Th -Ala - Leu - Lvs—Thr - A g-Thr—Gly- Sea: - Leu - Lys— Arg - Ala - Leu - His— Asn— Ala - Asp— Cys - Gin - Lys— Thr— Val- Thr— lie - Ser - Lys - Pro— Cys - Gly— Lys - Leu - Thr— Lys - Pro - Lys-Pro - Gin- Ala-Gl -Ser-Lys-Lys-Lys-Lys-Lys-Glu-Gly-Lys-Lys -Gln-Glu-Lys - Met - Leu— Asp 2) The protein according to claim 1, which is represented by the following sequence [I-I]: M t-Ser-Ser-Gln-Gln-Tyr-Gln-Gln-Gn-Arg-Arg-Lys-Phe-Ala-Ala-Ala— Phe-Leu— Ala-Leu-le-Phe—le- Leu— Ala— Ala-Val-Asp-Thi:-Ala- Gl -Ala-Gly-Lys-Lys-Glu-Lys-Pro-Glu-Lys-Lys-Val-Lys-Lys-Ser- Asp-Cys-Gly -Gl -Trp-Gln-Trp-Ser-Val-Cys-Val-Pro-Thr-Ser-Gly-Asp-Cys-Gly-Le -Gly-Thr-Arg-Glu-Gly-Thr-Arg-Thr-Gly -Ala-Glu- Cys— Iiys-Gin-Th-Met— Lys-Thi:-Gin— Arg-Cvs-Lys—elle-Pro-Cys— Asii- Trp-Lys-Lys-Gin— Phe-Gly-Ala -Glu-Cys-Iiys-Tyr-Gin-Phe-Gin-Ala-Trp-Glv-Glu-Cys-Asp-Leu-Asn-Th-Ala-Leu-Lvs-Thr-Ag-Thr-Gly- Sea: -Leu-Lys-Arg-Ala-Leu-His-Asn-Ala-Asp-Cys-Gin-Lys-Thr-Val-Thr-lie-Ser-Lys-Pro-Cys-Gly-Lys-Leu-Thr-Lys -Pro-Lys-Pro-Gin- Ala-Gl -Ser-Lys-Lys-Lys-Lys-Lys-Glu-Gly-Lys-Lys -Gln-Glu-Lys-Met-Leu— Asp 〔 I - I〕  [I-I] 3) 下記の配列 〔 1 — Π〕 で表される請求項 1記載の蛋白 質。 3) The protein according to claim 1, which is represented by the following sequence [1-Π]. Met - Gin - Ala - Gin - Gin— Tyr - Gin - G1II G1TI Arg- Arg - Lys - Phe - Ala - Ala - Ala— Phe - Leu - Ala - Phe -ェ le - Phe—ェ le - - Ala - Ala - Val— Asp - Thr - Ala - Glu - Ala - Gly- Lys - Lys- Glu- Lys - Pro - Glu - Lys- Lys -Val-Lys-Lys-Ser- Asp - Cys - Gly - Glu- Trp- Gin— Trp - Ser - Val— Cys - Val - Pro— Thr— Sear - Gly - Asp-Cys-Gly-Leu-Gly-Thr-Arg-Glu-Gly-Thr-Arg-Thr-Glv-Ala-Glu- Cvs - Lvs - Gin - Thr- Met - Lys - Thr— Gin- Arg - Cvs - L s -ェ le— Pro - Cys— Asn- Trp- Iiys- Lys- Gin- Phe - Gly- Ala - Glu - Cys - Lvs - Tyr - Gin - Phe- Gin- Ala - Trp - Gly - Glu— Cvs - Asp— Leu - Asn - Thr - Ala - Leu - Ijys— Thr - Arg— Thr - Gly— Ser-Leu - Lys— ArCT-Ala— Leu - His— Asn— AJLa— Glu-Cys - Gin - IJVS Thr— Val - Thr -ェ le - Sear— - Pro - Cys - Gly - Lys - Leu - Thr - Lys— Pro - Lys - Pro - Gin— Ala— Glu - Sei: - Lys - Lvs - Lys - Lvs - Lys - Glu - Gly - Lys - Lvs— Gin - GITI - Lys—
Figure imgf000027_0001
Met-Gin-Ala-Gin-Gin— Tyr-Gin-G1II G1TI Arg- Arg-Lys-Phe-Ala-Ala-Ala— Phe-Leu-Ala-Phe-e le-Phe — e le--Ala-Ala -Val— Asp-Thr-Ala-Glu-Ala-Gly- Lys-Lys- Glu- Lys-Pro-Glu-Lys- Lys -Val-Lys-Lys-Ser- Asp-Cys-Gly-Glu- Trp- Gin — Trp-Ser-Val— Cys-Val-Pro— Thr— Sear-Gly-Asp-Cys-Gly-Leu-Gly-Thr-Arg-Glu-Gly-Thr-Arg-Thr-Glv-Ala-Glu- Cvs -Lvs-Gin-Thr- Met-Lys-Thr— Gin- Arg-Cvs-Ls-cell— Pro-Cys— Asn- Trp- Iiys- Lys- Gin- Phe-Gly- Ala-Glu-Cys-Lvs -Tyr-Gin-Phe- Gin- Ala-Trp-Gly-Glu— Cvs-Asp— Leu-Asn-Thr-Ala-Leu-Ijys— Thr-Arg— Thr-Gly— Ser-Leu-Lys— ArCT-Ala — Leu-His— Asn— AJLa— Glu-Cys—Gin—IJVS Thr— Val—Thr—elle—Sear——Pro—Cys—Gly—Lys—Leu— Thr—Lys— Pro—Lys—Pro—Gin— Ala— Glu-Sei:-Lys-Lvs-Lys-Lvs-Lys-Glu-Gly-Lys-Lvs— Gin-GITI-Lys—
Figure imgf000027_0001
 〔 I - n〕  [I-n] 4) 請求項 1記載の配列 〔 I〕 の全体、 部分的に修飾され た配列の全体またはそれらの部分をコー ドする D N A。4) A DNA encoding the entire sequence [I] according to claim 1, the entire partially modified sequence or a portion thereof. 5) 下記の配列 〔 Π〕 からなる塩基配列の全体またはその 部分からなる請求項 4記載の D Ν Α。 5) The DΝII according to claim 4, which comprises the whole or a part of the nucleotide sequence consisting of the following sequence [Π]. ATG-YZG-XCY-CAU-CAU-TAY-CAG-CAG-CAU-CGT-ZGA-AAA-TTT-GCA-GCT- GCC-TTC-YTG-GCA-TTV-ATT-TTC-ATZ-YTG-GCA-GCT-GTG-GAY-ACT-GCT- GAU-GCZ-GGG-AAG-AAA-GAG-AAA-CC -GAA-AAA-AAU-GTG-AAU-AAG-TCT- GAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGY-GTG-CCY-ACC-AGY-GGU- GAC-TGT-GGU-YTG-GGC-ACZ-CGG-GAG-GGC-ACT-CGV-ACT-GGZ-GCY-GAG- TGC-AAU-CAU-ACC-ATG-AAG-ACY-CAG-AGA-TGT-AAG-ATC-CCY-TGC-AAC- TGG-AAG-AAG-CAU-TTT-GGZ-GCX-GAG-TGC-AAU-TAC-CAG-TTC-CAG-GCY- TGG-GGA-GAA-TGT-GAC-C V-AAY-ACZ-GCC-YTG-AAG-ACC-AGA-ACT-GGZ- AGY-CTG-AAG-CGA-GCY-CTG-C¾C-AAT-GCY-GAZ-TGY-CAG-AAU-ACT-GTC- ACC-ATC-TCC-AAG-CCC-TGT-GGC-AAU-C V-ACC-AAG-CCC-AAU-CCT-CAA- GCU - GAU - TCW - AAG - AAG-AAG - AAA - AAG - GAA - GGC-AAG - AAA-CAG-GAG-AAG - ATG-CTG-GAT-TAA ATG-YZG-XCY-CAU-CAU-TAY-CAG-CAG-CAU-CGT-ZGA-AAA-TTT-GCA-GCT- GCC-TTC-YTG-GCA-TTV-ATT-TTC-ATZ-YTG-GCA- GCT-GTG-GAY-ACT-GCT- GAU-GCZ-GGG-AAG-AAA-GAG-AAA-CC -GAA-AAA-AAU-GTG-AAU-AAG-TCT- GAC-TGT-GGA-GAA-TGG- CAG-TGG-AGT-GTG-TGY-GTG-CCY-ACC-AGY-GGU-GAC-TGT-GGU-YTG-GGC-ACZ-CGG-GAG-GGC-ACT-CGV-ACT-GGZ-GCY-GAG- TGC-AAU-CAU-ACC-ATG-AAG-ACY-CAG-AGA-TGT-AAG-ATC-CCY-TGC-AAC- TGG-AAG-AAG-CAU-TTT-GGZ-GCX-GAG-TGC-AAU- TAC-CAG-TTC-CAG-GCY- TGG-GGA-GAA-TGT-GAC-C V-AAY-ACZ-GCC-YTG-AAG-ACC-AGA-ACT-GGZ-AGY-CTG-AAG-CGA-GCY -CTG-C¾C-AAT-GCY-GAZ-TGY-CAG-AAU-ACT-GTC- ACC-ATC-TCC-AAG-CCC-TGT-GGC-AAU-C V-ACC-AAG-CCC-AAU-CCT- CAA- GCU-GAU-TCW-AAG-AAG-AAG-AAA-AAG-GAA-GGC-AAG-AAA-CAG-GAG-AAG-ATG-CTG-GAT-TAA 〔n〕 配列中、 Uは Aか G、 Yは Cか T、 Xは Gか T、 Zは A か C、 Vは Gか C、 Wは Tか Aである。  [N] In the sequence, U is A or G, Y is C or T, X is G or T, Z is A or C, V is G or C, and W is T or A. 6) 下記の配列 〔Π— I〕 からなる請求項 4記載の D N A 6) The DNA according to claim 4, which comprises the following sequence [Π-I]: ATG - TCG-TCC - CAG - - CAG - CAG - CAA-CGT - AGA - AAA - TTT - GCA - GCT -ATG-TCG-TCC-CAG--CAG-CAG-CAA-CGT-AGA-AAA-TTT-GCA-GCT- GCC-TTC-CTG-GCA-TTG-ATT-TTC-ATC-TTG-GCA-GCT-GTG-GAC-ACT-GCT-GCC-TTC-CTG-GCA-TTG-ATT-TTC-ATC-TTG-GCA-GCT-GTG-GAC-ACT-GCT- GAG-GCC-GGG-AAG-AAA-GAG-AAA-CCT-GAA-AAA-AAG-GTG-AAA-AAG-TCT-GAG-GCC-GGG-AAG-AAA-GAG-AAA-CCT-GAA-AAA-AAG-GTG-AAA-AAG-TCT- GAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGC-GTG-CCT-ACC-AGC-GGG-GAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGC-GTG-CCT-ACC-AGC-GGG- GAC-TGT-GGA-TTG-GGC-ACC-CGG-GAG-GGC-ACT-CGC-ACT-GGC-GCC-GAG-GAC-TGT-GGA-TTG-GGC-ACC-CGG-GAG-GGC-ACT-CGC-ACT-GGC-GCC-GAG- TGC - AAA - CAG - ACC— ATG - AAG - ACT—CAG-AGA_TGT_AAG_ATC_CCT-TGC_AAC_TGC-AAA-CAG-ACC— ATG-AAG-ACT—CAG-AGA_TGT_AAG_ATC_CCT-TGC_AAC_ TGG - AAG - AAG - CAG-TTT - GGA - GCT - GAG -: TGC - AAG - TAC - CAG-TTC-CAG - GCT -TGG-AAG-AAG-CAG-TTT-GGA-GCT-GAG-: TGC-AAG-TAC-CAG-TTC-CAG-GCT- TGG-GGA-GAA-TGT-GAC-CTC-AAT-ACC-GCC-TTG-AAG-ACC-AGA-ACT-GGC-TGG-GGA-GAA-TGT-GAC-CTC-AAT-ACC-GCC-TTG-AAG-ACC-AGA-ACT-GGC- AGC-CTG-AAG-CGA-GCT-CTG-CAC-AAT-GCT-GAC-TGT-CAG-AAA-ACT-GTC-AGC-CTG-AAG-CGA-GCT-CTG-CAC-AAT-GCT-GAC-TGT-CAG-AAA-ACT-GTC- ACC-ATC-TCC-AAG-CCC-TGT-GGC-AAG-CTC-ACC-AAG-CCC-AAG-CCT-CAA-ACC-ATC-TCC-AAG-CCC-TGT-GGC-AAG-CTC-ACC-AAG-CCC-AAG-CCT-CAA- GCG-GAG-TCA-AAG-AAG-AAG-AAA-AAG-GAA-GGC-AAG-AAA-CAG-GAG-AAG-GCG-GAG-TCA-AAG-AAG-AAG-AAA-AAG-GAA-GGC-AAG-AAA-CAG-GAG-AAG- ATG-CTG-GAT-TAA ATG-CTG-GAT-TAA 〔n— I〕  [N-I] 7) 下記の配列 〔H— Π〕 からなる請求項 4記載の D N A 7) The DNA according to claim 4, comprising the following sequence [H-Π]: ATG-CAG - GCT - CAA - CAG - TAC - CAG - CAG-CAG - CGT - CGA - AAA - TTT - GCA - GCT - GCC-TTC-TTG-GCA-TTC-ATT-TTC-ATA-CTG-GCA-GCT-GTG-GAT-ACT-GCT- GAA-GCA-GGG-AAG-AAA-GAG-AAA-CCA-GAA-AAA-AAA-GTG-AAG-AAG-TCT- GAC-TGT-GGA-GAA-TGG-CAG-TGG-AGT-GTG-TGT-GTG-CCC-ACC-AGT-GGA- GAC-TGT-GGG-CTG-GGC-ACA-CGG-GAG-GGC-ACT-CGG-ACT-GGA-GCT-GAG- TGC - AAG - CAA-ACC- ATG-AAG - ACC - CAG-AGA— TGT - AAG-ATC-CCC - TGC-AAC- TGG - AAG - AAG - CAA- !TTT - GGC - GCG - GAG - TGC - AAA- TAC - CAG - TTC - CAG - GCC - TGG-GGA-GAA-TGT-GAC-CTG-AAC-ACA-GCC-CTG-AAG-ACC-AGA-ACT-GGA- AGT-CTG-AAG-CGA-GCC-CTG-CAC-AAT-GCC-GAA-TGC-CAG-AAG-ACT-GTC- ACC-ATC-TCC-AAG-CCC-TGT-GGC-AAA-CTG-ACC-AAG-CCC-AAA-CCT-CAA- GCA-GAA-TCT-AAG-AAG-AAG-AAA-AAG-GAA-GGC-AAG-AAA-CAG-GAG-AAG- ATG-CTG-GAT-TAA ATG-CAG-GCT-CAA-CAG-TAC-CAG-CAG-CAG-CGT-CGA-AAA-TTT-GCA-GCT-GCC-TTC-TTG-GCA-TTC-ATT-TTC-ATA-CTG-GCA- GCT-GTG-GAT-ACT-GCT- GAA-GCA-GGG-AAG-AAA-GAG-AAA-CCA-GAA-AAA-AAA-GTG-AAG-AAG-TCT-GAC-TGT-GGA-GAA-TGG- CAG-TGG-AGT-GTG-TGT-GTG-CCC-ACC-AGT-GGA-GAC-TGT-GGG-CTG-GGC-ACA-CGG-GAG-GGC-ACT-CGG-ACT-GGA-GCT-GAG- TGC-AAG-CAA-ACC- ATG-AAG-ACC-CAG-AGA— TGT-AAG-ATC-CCC-TGC-AAC- TGG-AAG-AAG-CAA-! TTT-GGC-GCG-GAG-TGC-AAA -TAC-CAG-TTC-CAG-GCC-TGG-GGA-GAA-TGT-GAC-CTG-AAC-ACA-GCC-CTG-AAG-ACC-AGA-ACT-GGA- AGT-CTG-AAG-CGA-GCC -CTG-CAC-AAT-GCC-GAA-TGC-CAG-AAG-ACT-GTC-ACC-ATC-TCC-AAG-CCC-TGT-GGC-AAA-CTG-ACC-AAG-CCC-AAA-CCT-CAA -GCA-GAA-TCT-AAG-AAG-AAG-AAA-AAG-GAA-GGC-AAG-AAA-CAG-GAG-AAG- ATG-CTG-GAT-TAA 〔Π—！！〕 8) 請求項 1記載の蛋白質をコー ドする D N A配列を有す るプラス ミ ドベク タ一。  [Π—! ! [8] A plasmid having a DNA sequence encoding the protein according to [1]. 9) 請求項 1記載の蛋白質をコー ドする D N A配列を有す る発現べク タ一で形質転換した宿主を培養することを特 徴とする請求項 1記載の蛋白質を生産する方法。  9) A method for producing the protein according to claim 1, wherein the host is transformed with an expression vector having a DNA sequence encoding the protein according to claim 1. 1 0 ) 請求項 1乃至 3のいずれかの項に記載の蛋白質および 製薬的に許容しう る担体または希釈剤を含む医薬。  10) A medicament comprising the protein according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or diluent.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0562508A3 (en) * 1992-03-27 1994-06-22 Hoechst Japan Protein with bone formation ability and process for its production
EP0585801A3 (en) * 1992-08-28 1994-06-29 Hoechst Japan Bone-related cadherin-like protein and process for its production
WO1999034820A1 (en) * 1998-01-09 1999-07-15 Heikki Matti Eemeli Rauvala New use of heparin-binding growth-associated molecule
WO2005014022A1 (en) 2003-07-16 2005-02-17 Develogen Aktiengesellschaft Use of pleitrophin for preventing and treating pancreatic diseases and/or obesity and/or metabolic syndrome
KR100554965B1 (en) * 2003-04-23 2006-03-03 주식회사 남승 Core Spandex Dyed Yarn for Jeans and Manufacturing Method Thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol. 151, No. 3, (1988), K. KADOMATSU et al., "Complementary DNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis", p. 1312-1318. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 265, No. 16, (1990), S. MATSUBARA et al., "Structure of a Retinoic Acidresponsive gene, MK, which is transiently Activated during the differentiation of embryonal carcinoma cells and the midgestation period of mouse embryogenesis", p. 9441-9443. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 265, No. 18, (1990), M. TOMOMURA et al., "A retinoic acid-responsive Gene, MK, found in the teratocarcinoma", p. 10765-10770. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0562508A3 (en) * 1992-03-27 1994-06-22 Hoechst Japan Protein with bone formation ability and process for its production
EP0585801A3 (en) * 1992-08-28 1994-06-29 Hoechst Japan Bone-related cadherin-like protein and process for its production
US5869638A (en) * 1992-08-28 1999-02-09 Hoechst Japan Limited Bone-related cadherin-like protein and process for its production
WO1999034820A1 (en) * 1998-01-09 1999-07-15 Heikki Matti Eemeli Rauvala New use of heparin-binding growth-associated molecule
KR100554965B1 (en) * 2003-04-23 2006-03-03 주식회사 남승 Core Spandex Dyed Yarn for Jeans and Manufacturing Method Thereof
WO2005014022A1 (en) 2003-07-16 2005-02-17 Develogen Aktiengesellschaft Use of pleitrophin for preventing and treating pancreatic diseases and/or obesity and/or metabolic syndrome

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